Antisense antibacterial compounds and methods

ABSTRACT

Provided are antisense morpholino oligomers targeted against bacterial virulence factors such as genes that contribute to antibiotic resistance or biofilm formation, or genes associated with fatty acid biosynthesis, and related compositions and methods of using the oligomers and compositions, for instance, in the treatment of an infected mammalian subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Application No. 61/994,750, filed May 16, 2014, U.S. Application No.62/099,046, filed Dec. 31, 2014, and U.S. Application No. 62/129,746,filed Mar. 6, 2015; each of which is incorporated by reference in itsentirety.

STATEMENT REGARDING THE SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is SATH-003_03US_ST25.txt. The text file is about10 KB, was created on May 15, 2015, and is being submittedelectronically via EFS-Web.

BACKGROUND

Technical Field

The present disclosure relates to antisense morpholino oligomerstargeted against bacterial virulence factors such as genes thatcontribute to antibiotic resistance, biofilm formation or fatty acidbiosynthesis, and related compositions and methods of using theoligomers and compositions, for instance, in the treatment of aninfected mammalian subject.

Description of the Related Art

Currently, there are several types of antibiotic compounds in useagainst bacterial pathogens and these compounds act through a variety ofanti-bacterial mechanisms. For example, beta-lactam antibiotics, such aspenicillin and cephalosporin, act to inhibit the final step inpeptidoglycan synthesis. Glycopeptide antibiotics, including vancomycinand teichoplanin, inhibit both transglycosylation and transpeptidationof muramyl-pentapeptide, again interfering with peptidoglycan synthesis.Other well-known antibiotics include the quinolones, which inhibitbacterial DNA replication, inhibitors of bacterial RNA polymerase, suchas rifampin, and inhibitors of enzymes in the pathway for production oftetrahydrofolate, including the sulfonamides.

Some classes of antibiotics act at the level of protein synthesis.Notable among these are the aminoglycosides, such as kanamycin andgentamicin. This class of compounds targets the bacterial 30S ribosomesubunit, preventing the association with the 50S subunit to formfunctional ribosomes. Tetracyclines, another important class ofantibiotics, also target the 30S ribosome subunit, acting by preventingalignment of aminoacylated tRNA's with the corresponding mRNA codon.Macrolides and lincosamides, another class of antibiotics, inhibitbacterial synthesis by binding to the 50S ribosome subunit, andinhibiting peptide elongation or preventing ribosome translocation.

Despite impressive successes in controlling or eliminating bacterialinfections by antibiotics, the widespread use of antibiotics both inhuman medicine and as a feed supplement in poultry and livestockproduction has led to drug resistance in many pathogenic bacteria.Antibiotic resistance mechanisms can take a variety of forms. One of themajor mechanisms of resistance to beta lactams, particularly inGram-negative bacteria, is the enzyme beta-lactamase, which renders theantibiotic inactive by cleaving the lactam ring. Likewise, resistance toaminoglycosides often involves an enzyme capable of inactivating theantibiotic, in this case by adding a phosphoryl, adenyl, or acetylgroup. Active efflux of antibiotics is another way that many bacteriadevelop resistance. Genes encoding efflux proteins, such as the tetA,tetG, tetL, and tetK genes for tetracycline efflux, have beenidentified. A bacterial target may develop resistance by altering thetarget of the drug. For example, the so-called penicillin bindingproteins (PBPs) in many beta-lactam resistant bacteria are altered toinhibit the critical antibiotic binding to the target protein.Resistance to tetracycline may involve, in addition to enhanced efflux,the appearance of cytoplasmic proteins capable of competing withribosomes for binding to the antibiotic. For those antibiotics that actby inhibiting a bacterial enzyme, such as for sulfonamides, pointmutations in the target enzyme may confer resistance.

Biofilm formation can also lead to antibiotic resistance, among otherclinical difficulties. Typically, in situations where bacteria form abiofilm within an infected host, the infection turns out to beuntreatable and can develop into a chronic state. Hallmarks of chronicbiofilm-based infections not only include resistance to antibiotictreatments and many other conventional antimicrobial agents but also acapacity for evading host defenses. Therefore, strategies that preventor breakdown biofilm would be of therapeutic interest and benefit.

The appearance of antibiotic resistance in many pathogenic bacteria,including cases involving multi-drug resistance (MDR), raises the fearof a post-antibiotic era in which many bacterial pathogens were simplyuntreatable by medical intervention. Thus, there is a need forantimicrobial agents that (i) are not subject to the principal types ofantibiotic resistance currently hampering antibiotic treatment ofbacterial infection, (ii) can be developed rapidly and with somereasonable degree of predictability as to target-bacteria specificity,(iii) are effective at low doses, and (iv) show few side effects.

SUMMARY

Embodiments of the present disclosure relate, in part, to the discoverythat the antisense targeting of bacterial virulence factors can, interalia, increase the antibiotic susceptibility of otherwiseantibiotic-resistant pathogenic bacteria, and reduce the ability ofcertain pathogenic bacteria to form and maintain difficult-to-treatbiofilms. For example, the antisense targeting of antibiotic resistancegenes such as carbapenemases and efflux pumps was shown to increase thesusceptibility of antibiotic resistant (e.g., multi-drug resistant)bacteria to many commonly used antibiotics, and could thus find utilityin the treatment of such bacteria, for instance, in combination withantibiotics. Also, the antisense targeting of genes associated withbiofilm formation was shown to break down existing biofilms and reducethe production of new biofilms. Such antisense targeting could findutility in standalone therapies against biofilm-forming bacteria, and ascombination therapies, for example, to increase the susceptibility ofbiofilm-forming bacteria to antibiotics.

Embodiments of the present disclosure therefore include a substantiallyuncharged antisense morpholino oligomer, composed of morpholino subunitsand phosphorus-containing intersubunit linkages joining a morpholinonitrogen of one subunit to a 5′-exocyclic carbon of an adjacent subunit,and having (a) about 10-40 nucleotide bases, and (b) a targetingsequence of sufficient length and complementarity to specificallyhybridize to a bacterial mRNA target sequence that encodes a virulencefactor; where the oligomer is conjugated to a cell-penetrating peptide(CPP).

In certain embodiments, the target sequence comprises a translationalstart codon of the bacterial mRNA and/or a sequence within about 30bases upstream or downstream of the translational start codon of thebacterial mRNA.

In some embodiments, the virulence factor is an antibiotic resistanceprotein, a biofilm formation protein or a protein associated with fattyacid biosynthesis.

In certain embodiments, the antibiotic resistance protein is selectedfrom one or more of New Delhi metallo-beta-lactamase (NDM-1) andresistance-nodulation-cell division (RND)-type multidrug efflux pumpsubunit AdeA (adeA). In specific embodiments, the target sequence isselected from Table 1A. Some antisense oligomers comprise, consist, orconsist essentially of a targeting sequence set forth in Table 2A, afragment of at least 10 contiguous nucleotides of a targeting sequencein Table 2A, or variant having at least 80% sequence identity to atargeting sequence in Table 2A.

In some embodiments, the biofilm formation protein is encoded by one ormore of cepI or suhB. In particular embodiments, the target sequence isselected from Table 1B. Some antisense oligomers comprise, consist, orconsist essentially of a targeting sequence set forth in Table 2B, afragment of at least 10 contiguous nucleotides of a targeting sequencein Table 2B, or variant having at least 80% sequence identity to atargeting sequence in Table 2B.

In some embodiments, the protein associated with fatty acid biosynthesisis an acyl carrier protein encoded by one or more of acpP. In particularembodiments, the target sequence is selected from Table 1C. Someantisense oligomers comprise, consist, or consist essentially of atargeting sequence set forth in Table 2C, a fragment of at least 10contiguous nucleotides of a targeting sequence in Table 2C, or varianthaving at least 80% sequence identity to a targeting sequence in Table2C.

In certain embodiments, an antisense morpholino oligomer of thedisclosure may be of formula (I):

or a pharmaceutically acceptable salt thereof,

where each Nu is a nucleobase which taken together forms a targetingsequence;

X is an integer from 9 to 38;

T is selected from OH and a moiety of the formula:

where each R⁴ is independently C₁-C₆ alkyl, and R⁵ is selected from anelectron pair and H, and R⁶ is selected from OH, —N(R⁷)CH₂C(O)NH₂, and amoiety of the formula:

where:

-   -   R⁷ is selected from H and C₁-C₆ alkyl, and    -   R⁸ is selected from G, —C(O)—R⁹OH, acyl, trityl, and        4-methoxytrityl, where:        -   R⁹ is of the formula —(O-alkyl)_(y)- wherein y is an integer            from 3 to 10 and each of the y alkyl groups is independently            selected from C₂-C₆ alkyl;    -   each instance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is        independently C₁-C₆ alkyl, and R¹¹ is selected from an electron        pair and H;    -   R² is selected from H, G, acyl, trityl, 4-methoxytrityl,        benzoyl, stearoyl, and a moiety of the formula:

where L is selected from —C(O)(CH₂)₆C(O)— and —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,and each R¹² is of the formula —(CH₂)₂OC(O)N(R¹⁴)₂ wherein each R¹⁴ isof the formula —(CH₂)₆NHC(═NH)NH₂; and

R³ is selected from an electron pair, H, and C₁-C₆ alkyl,

wherein G is a cell penetrating peptide (“CPP”) and linker moietyselected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,—C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP,

and —C(O)CH₂NH—CPP, or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus, with the proviso that only one instance of G ispresent,

wherein the targeting sequence specifically hybridizes to a bacterialmRNA target sequence that encodes a virulence factor.

In certain embodiments, the CPP is an arginine-rich peptide. In certainembodiments, the CPP is selected from Table 1C.

Also included are methods of reducing expression and activity of avirulence factor in a bacteria or bacterium, comprising contacting thebacteria or bacterium with an antisense oligomer described herein.

In some embodiments, the bacterium is in a subject, and the methodcomprises administering the antisense oligomer to the subject.

In certain embodiments, the bacterium is selected from the genusEscherichia, Acinetobacter, Klebsiella, and Burkholderia. In certainembodiments, the bacterium is Escherichia coli, Acinetobacter baumannii,Klebsiella pneumoniae, or Burkholderia cepacia (complex). In certainembodiments, the bacterium is Escherichia coli, Acinetobacter baumannii,or Klebsiella pneumoniae, and where the virulence factor is anantibiotic resistance protein selected from one or more of NDM-1 andAdeA.

In some embodiments, the bacterium is Burkholderia cepacia (complex) andwhere the virulence factor is a biofilm formation protein encoded by oneor more of cepI and suhB. In certain embodiments, the Burkholderiacepacia (complex) comprises one or more of Burkholderia cenocepacia,Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderiastabilis, Burkholderia anthina, Burkholderia pyrrocinia, Burkholderiadolosa, and/or Burkholderia ambifaria. In certain embodiments,administration of the antisense oligomer reduces biofilm formation orexisting biofilm by at least about 10%. In certain embodiments, thesubject is immunocompromised and has an underlying lung disease. Inspecific embodiments, the subject has cystic fibrosis (CF) or chronicgranulomatous disease (CGD).

In some embodiments, the bacterium is Burkholderia cepacia (complex) andwhere the virulence factor is an acyl carrier protein associated withfatty acid biosynthesis encoded by one or more of acpP. In certainembodiments, the Burkholderia cepacia (complex) comprises one or more ofBurkholderia cenocepacia, Burkholderia multivorans, Burkholderiavietnamiensis, Burkholderia stabilis, Burkholderia anthina, Burkholderiapyrrocinia, Burkholderia dolosa, and/or Burkholderia ambifaria. Incertain embodiments, administration of the antisense oligomer reducesbiofilm formation or existing biofilm by at least about 10%. In certainembodiments, the subject is immunocompromised and has an underlying lungdisease. In specific embodiments, the subject has cystic fibrosis (CF)or chronic granulomatous disease (CGD).

Some methods include administering the oligomer separately orconcurrently with an antimicrobial agent, for example, whereadministration of the oligomer increases susceptibility of the bacteriumto the antimicrobial agent. Some methods include administering theoligomer by aerosolization.

In certain embodiments, the bacterium is Escherichia coli, Acinetobacterbaumannii, or Klebsiella pneumoniae, the virulence factor is NDM-1, andthe antimicrobial agent is a carbapenem. In certain embodiments, thecarbapenem is selected from one or more of meropenem, imipenem,ertapenem, doripenem, panipenem, biapenem, razupenem, tebipenem,lenapenem, and tomopenem.

In some embodiments, the bacterium is Escherichia coli, Acinetobacterbaumannii, or Klebsiella pneumoniae, the virulence factor is AdeA, andthe antimicrobial agent is selected from one or more of aminoglycosideantibiotics, tetracycline antibiotics, and β-lactam antibiotics. Incertain embodiments, the aminoglycoside is selected from one or more oftobramycin, gentamicin, kanamycin a, amikacin, dibekacin, sisomicin,netilmicin, neomycin B, neomycin C, neomycin E (paromomycin), andstreptomycin. In certain embodiments, the tetracycline antibiotic isselected from one or more of tetracycline, chlortetracycline,oxytetracycline, demeclocycline, lymecycline, meclocycline,methacycline, minocycline, rolitetracycline, and doxycyline. In certainembodiments, the β-lactam antibiotic is selected from one or more ofcarbapenems, penicillin derivatives (penams), cephalosporins (cephems),and monobactams.

In certain embodiments, the bacterium is Burkholderia cepacia (complex),the virulence factor is a biofilm formation protein encoded by one ormore of cepI or suhB, and the antimicrobial agent is selected from oneor more of ceftazidime, doxycycline, piperacillin, meropenem,chloramphenicol, and co-trimoxazole (trimethoprim/sulfamethoxazole).

In certain embodiments, the bacterium is Burkholderia cepacia (complex),the virulence factor is an acyl carrier protein associated with fattyacid biosynthesis encoded by one or more of acpP, and the antimicrobialagent is selected from one or more of ceftazidime, minocycline,doxycycline, piperacillin, meropenem, chloramphenicol, andco-trimoxazole (trimethoprim/sulfamethoxazole).

In some embodiments, the oligomer reduces the minimum inhibitoryconcentration (MIC) of the antimicrobial agent against the bacterium byat least about 10% relative to the antimicrobial agent alone. In certainembodiments, the oligomer increases the susceptibility of the bacteriumto the antimicrobial agent by at least about 10% relative to theantimicrobial agent alone.

Also included are pharmaceutical compositions, comprising an antisenseoligomer described herein and a pharmaceutically-acceptable carrier.Certain pharmaceutical compositions can further comprise one or moreantimicrobial agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an exemplary morpholino oligomer structure with aphosphorodiamidate linkage. FIGS. 1B-E show the repeating subunitsegment of exemplary morpholino oligomers, designated B through E. FIGS.1F-H show exemplary peptide PMO conjugates structures used in theexemplary PPMOs.

FIG. 2 shows that treatment of AdeA (efflux pump)-expressingAcinetobacter baumanii with a PPMO targeted against adeA significantlyreduced the MIC of the aminoglycoside antibiotic gentamicin.

FIG. 3 shows that treatment of AdeA-expressing Acinetobacter baumaniiwith a PPMO targeted against adeA significantly reduced the MIC of theaminoglycoside antibiotic tobramycin.

FIG. 4 shows that treatment of AdeA-expressing Acinetobacter baumaniiwith a PPMO targeted against adeA significantly reduced the MIC oftetracycline.

FIG. 5A shows that treatment of NDM-1-expressing Acinetobacter baumaniiwith a PPMO targeted against NDM-1 significantly reduced the MIC of thecarbapenem antibiotic meropenem. FIG. 5B shows that the NDM-1 targetedPPMO and meropenem synergistically reduced the number of colony-formingunits (CFUs) of NDM-1-expressing Acinetobacter baumanii.

FIG. 6 shows that treatment of NDM-1-expressing Escherichia coli with aPPMO targeted against NDM-1 significantly reduced the MIC of thecarbapenem antibiotic meropenem.

FIGS. 7A-7B show that treatment of biofilm-forming Burkholderia withPPMOs targeted against acpP, suhB or cepI not only disrupted theformation of biofilm (7A; PPMOs were added prior to biofilm formationand incubated for 48 hours) but also broke down established biofilms(7B; biofilm was grown for 48 hours prior to 48-hour incubation withPPMOs).

FIGS. 8A-8C visually demonstrate the reduction of biofilm formation onMBEC pegs using fluorescent-expressing Burkholderia and utilizingconfocal microscopy. FIG. 8A shows an untreated biofilm at 48 hours,FIG. 8B shows the 48-hour biofilm treated with 10 μM of the scramblePPMO control, and FIG. 8C shows the 48-hour biofilm treated with 10 μMof a cepI-targeted PPMO).

FIG. 9 shows that cepI PPMO and the aminoglycoside antibiotic Tobramycinare synergistic in reducing biofilm organism burden.

FIG. 10 shows a heat map of the minimal inhibitory concentration (MIC)values for various PPMOs including ones listed in Table 2C. The PPMOswere tested against a panel of 39 Bcc clinical isolates with varyinglevels of antibiotic resistance.

FIG. 11 shows that PPMOs are bactericidal in Bcc. Two different isolatesof B. cenocepacia were incubated for 24 hours in the presence or absenceof different acpP PPMOs. All three acpP PPMOs caused a significantreduction of growth in the clinical CF isolate B. cenocepacia K56-2(Panel A) and this effect was also seen for the pan-resistant strain B.cenocepacia HI4277 (Panel B).

FIG. 12 shows that acpP PPMO inhibits Bcc growth in artificial CFsputum. B. cenocepacia K56-2 was incubated alone or in the presence ofeither a scrambled sequence (Scr) placebo PPMO or acpP PPMO (PPMO#15,Table 2C) at 10 μM or 20 μM concentration. Media or PPMO was dosed at 2,8 and 12 hours. Samples were plated at 24 hours and CFU/ml wasdetermined.

FIG. 13 shows that acpP PPMO prevents biofilm formation in B.cenocepacia J2315. B. cenocepacia J2315 was grown utilizing MBEC biofilmassay plates for 48 hours in the presence of either acpP PPMO (10 μM),scrambled PPMO (10 μM), peptide or media alone. Biofilm production wasmeasure utilizing a crystal violet method.

FIGS. 14A-14C show that acpP PPMO can break down an existing B.cenocepacia biofilm. dsRed-expressing B. cenocepacia J2315 was grown onMBEC pegs for 48 hours. The pegs were moved to fresh media containingeither nothing, the Scrambled (Scr) control PPMO at 10 μM concentration,or the acpP PPMO at 10 μM concentration. The MBEC pegs were incubatedfor another 48 hours and then stained with fluorescent greenpeanut-agglutinin stain for the biofilm and imaged on confocalmicroscopy. While no PPMO (FIG. 14A) or Scr PPMO (FIG. 14B) displayedthick biofilms, the acpP PPMO-treated pegs (FIG. 14C) showed biofilmwith pockets of no visible organisms.

FIG. 15 shows that aerosol delivery of PPMO reduces the burden of B.multivorans in a pulmonary infection model. Chronic granulomatousdisease (CGD) mice were used as a Bcc infection model. An Aerogennebulizer was used to deliver either scrambled (Scr) PPMO (300 μg) oracpP PPMO (PPMO#15, Table 2C, 300 μg or 30 μg) as a one-time dose 6hours post-infection. Mice were euthanized 24 hours after infection andlung burden was determined as CFU/g.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, preferred methods andmaterials are described. For the purposes of the present disclosure, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight, or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight, or length.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notdirectly contribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of:” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

As used herein, the terms “contacting a cell”, “introducing” or“delivering” include delivery of the oligomers of this disclosure into acell by methods routine in the art, e.g., transfection (e.g., liposome,calcium-phosphate, polyethyleneimine), electroporation (e.g.,nucleofection), microinjection), transformation, and administration.

The terms “cell penetrating peptide” (CPP) or “a peptide moiety whichenhances cellular uptake” are used interchangeably and refer to cationiccell penetrating peptides, also called “transport peptides”, “carrierpeptides”, or “peptide transduction domains”. In some aspects, thepeptides have the capability of inducing cell penetration within aboutor at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells ofa given population and/or allow macromolecular translocation to orwithin multiple tissues in vivo upon systemic administration. Particularexamples of CPPs include “arginine-rich peptides.” CPPs are well-knownin the art and are disclosed, for example, in U.S. Application No.2010/0016215, which is incorporated by reference in its entirety.

“An electron pair” refers to a valence pair of electrons that are notbonded or shared with other atoms.

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395) or BLAST. In this waysequences of a similar or substantially different length to those citedherein could be compared by insertion of gaps into the alignment, suchgaps being determined, for example, by the comparison algorithm used byGAP.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide” or “isolated oligomer,” as usedherein, may refer to a polynucleotide that has been purified or removedfrom the sequences that flank it in a naturally-occurring state, e.g., aDNA fragment that is removed from the sequences that are adjacent to thefragment in the genome. The term “isolating” as it relates to cellsrefers to the purification of cells (e.g., fibroblasts, lymphoblasts)from a source subject (e.g., a subject with a polynucleotide repeatdisease). In the context of mRNA or protein, “isolating” refers to therecovery of mRNA or protein from a source, e.g., cells.

The term “modulate” includes to “increase” or “decrease” one or morequantifiable parameters, optionally by a defined and/or statisticallysignificant amount. By “increase” or “increasing,” “enhance” or“enhancing,” or “stimulate” or “stimulating,” refers generally to theability of one or antisense compounds or compositions to produce orcause a greater physiological response (i.e., downstream effects) in acell or a subject relative to the response caused by either no antisensecompound or a control compound. Relevant physiological or cellularresponses (in vivo or in vitro) will be apparent to persons skilled inthe art. An “increased” or “enhanced” amount is typically a“statistically significant” amount, and may include an increase that is1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times(e.g., 500, 1000 times) (including all integers and ranges between andabove 1), e.g., 1.5, 1.6, 1.7, 1.8) the amount produced by no antisensecompound (the absence of an agent) or a control compound. The term“reduce” or “inhibit” may relate generally to the ability of one or moreantisense compounds or compositions to “decrease” a relevantphysiological or cellular response, such as a symptom of a disease orcondition described herein, as measured according to routine techniquesin the diagnostic art. Relevant physiological or cellular responses (invivo or in vitro) will be apparent to persons skilled in the art, andmay include reductions in bacterial cell growth, reductions in theminimum inhibitory concentration (MIC) of an antimicrobial agent, andothers. A “decrease” in a response may be “statistically significant” ascompared to the response produced by no antisense compound or a controlcomposition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease,including all integers and ranges in between.

As used herein, an “antisense oligomer,” “oligomer” or “oligomer” refersto a linear sequence of nucleotides, or nucleotide analogs, which allowsthe nucleobase (for example a purine or pyrimidine base-pairing moiety)to hybridize to a target sequence in an RNA by Watson-Crick basepairing, to form an oligomer:RNA heteroduplex within the targetsequence. The terms “antisense oligomer”, “antisense oligomer”,“oligomer” and “compound” may be used interchangeably to refer to anoligomer. The cyclic subunits may be based on ribose or another pentosesugar or, in certain embodiments, a morpholino group (see description ofmorpholino oligomers below).

The term “oligomer,” “oligomer,” or “antisense oligomer” alsoencompasses an oligomer having one or more additional moietiesconjugated to the oligomer, e.g., at its 3′- or 5′-end, such as apolyethylene glycol moiety or other hydrophilic polymer, e.g., onehaving 10-100 monomeric subunits, which may be useful in enhancingsolubility, or a moiety such as a lipid or peptide moiety that iseffective to enhance the uptake of the compound into target bacterialcells and/or enhance the activity of the compound within the cell, e.g.,enhance its binding to a target polynucleotide.

A “nuclease-resistant” oligomers refers to one whose backbone issubstantially resistant to nuclease cleavage, in non-hybridized orhybridized form; by common extracellular and intracellular nucleases inthe body or in a bacterial cell (for example, by exonucleases such as3′-exonucleases, endonucleases, RNase H); that is, the oligomer showslittle or no nuclease cleavage under normal nuclease conditions to whichthe oligomer is exposed. A “nuclease-resistant heteroduplex” refers to aheteroduplex formed by the binding of an antisense oligomer to itscomplementary target, such that the heteroduplex is substantiallyresistant to in vivo degradation by intracellular and extracellularnucleases, which are capable of cutting double-stranded RNA/RNA orRNA/DNA complexes. A “heteroduplex” refers to a duplex between anantisense oligomer and the complementary portion of a target RNA.

As used herein, “nucleobase” (Nu), “base pairing moiety” or “base” areused interchangeably to refer to a purine or pyrimidine base found innative DNA or RNA (uracil, thymine, adenine, cytosine, and guanine), aswell as analogs of the naturally occurring purines and pyrimidines, thatconfer improved properties, such as binding affinity to the oligomer.Exemplary analogs include hypoxanthine (the base component of thenucleoside inosine); 2,6-diaminopurine; 5-methyl cytosine;C5-propynyl-modified pyrimidines; 9-(aminoethoxy)phenoxazine (G-clamp)and the like.

A nucleobase covalently linked to a ribose, sugar analog or morpholinocomprises a nucleoside. “Nucleotides” are composed of a nucleosidetogether with one phosphate group. The phosphate groups covalently linkadjacent nucleotides to one another to form an oligomer.

An oligomer “specifically hybridizes” to a target sequence if theoligomer hybridizes to the target under physiological conditions, with aTm substantially greater than 40° C. or 45° C., preferably at least 50°C., and typically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

As used herein, “sufficient length” includes an antisense oligomer thatis complementary to at least about 8, more typically about 8-10, 8-11,8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-30, 8-40, or10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20,10-30, 10-40 (including all integers and ranges in between) contiguousor non-contiguous nucleobases in a region of a bacterial mRNA targetsequence. An antisense oligomer of sufficient length has at least aminimal number of nucleotides to be capable of specifically hybridizingto a region of the bacterial mRNA target. In some embodiments, anoligomer of sufficient length is from 10 to 40 or 10 to 30 nucleotidesin length, for example, about 10-11, 10-12, 10-13, 10-14, 10-15, 10-16,10-17, 10-18, 10-19, 10-20, 10-25, 10-28, 10-30, 10-40, 11-12, 11-13,11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-25, 11-28, 11-30, or11-40 nucleotides in length, or about 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40 nucleotides in length.

The terms “sequence identity” or, for example, comprising a “sequence50% identical to,” as used herein, refer to the extent that sequencesare identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e., resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.,Nucl. Acids Res. 25:3389, 1997.

A “subject” or a “subject in need thereof” includes a mammalian subjectsuch as a human subject.

The terms “TEG,” “EG3,” or “triethylene glycol tail” refer totriethylene glycol moieties conjugated to the oligomer, e.g., at its 3′-or 5′-end. For example, in some embodiments, “TEG” includes, forexample, wherein T of the compound of formula (I), (II), or (III) is ofthe formula:

The term “pip-PDA” refers to a 5′ terminal piperazine-phosphorodiamidatemoiety that connects a G group, where the G group comprises acell-penetrating peptide (CPP) and linker moiety further discussedbelow, to the 5′ end of the oligomer by way of an amide bond between theG group linker and the piperazinyl nitrogen. For example, in someembodiments, “pip-PDA” includes wherein T of the compound of formula (I)or (II) is of the formula:

The term “target sequence” refers to a portion of the target RNA, forexample, a bacterial mRNA, against which the antisense oligomer isdirected, that is, the sequence to which the oligomer will hybridize byWatson-Crick base pairing of a complementary sequence. In certainembodiments, the target sequence may be a contiguous region of thetranslation initiation region of a bacterial gene.

The “translational start codon region” refers to a region that is 30bases upstream or downstream of a translation initiation codon of agene.

The term “targeting sequence” or “antisense targeting sequence” refersto the sequence in an oligomer that is complementary or substantiallycomplementary to the target sequence in the RNA, e.g., the bacterialmRNA. The entire sequence, or only a portion, of the antisense compoundmay be complementary to the target sequence. For example, in an oligomerof about 10-30 bases, about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of the bases may betargeting sequences that are complementary to the target region.Typically, the targeting sequence is formed of contiguous bases, but mayalternatively be formed of non-contiguous sequences that when placedtogether, e.g., from opposite ends of the oligomer, constitute sequencethat spans the target sequence.

A “targeting sequence” may have “near” or “substantial” complementarityto the target sequence and still function for the purpose of the presentdisclosure, that is, still be “complementary.” Preferably, the oligomeranalog compounds employed in the present disclosure have at most onemismatch with the target sequence out of 10 nucleotides, and preferablyat most one mismatch out of 20. Alternatively, the antisense oligomersemployed have at least 90% sequence homology, and preferably at least95% sequence homology, with the exemplary targeting sequences asdesignated herein.

As used herein, the term “quantifying”, “quantification” or otherrelated words refer to determining the quantity, mass, or concentrationin a unit volume, of a nucleic acid, polynucleotide, oligomer, peptide,polypeptide, or protein.

As used herein, “treatment” of a subject (e.g. a mammal, such as ahuman) or a cell is any type of intervention used in an attempt to alterthe natural course of the individual or cell. Treatment includes, but isnot limited to, administration of a pharmaceutical composition, and maybe performed either prophylactically or subsequent to the initiation ofa pathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition,or associated symptoms thereof.

Sequences for Targeting Bacterial Virulence Factors

Certain embodiments relate to antisense oligomers, and relatedcompositions and methods, which are of sufficient length andcomplementarity to specifically hybridize to a bacterial mRNA targetsequence that encodes a virulence factor. General examples of virulencefactors include antibiotic resistance genes, biofilm formation genes,genes associated with fatty acid biosynthesis and their encodedproteins. In addition, virulence factors include genes that encoderegulatory proteins that control the expression (transcription and/ortranslation) of other genes which provide a benefit to the bacteriumduring the process of infection.

In certain embodiments, the target sequence contains all or a portion(e.g., 1 or 2 nucleotides) of a translational start codon of thebacterial mRNA. In some embodiments, the target sequence contains asequence that is about or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 bases upstream or downstream of a translational start codon(e.g., ATG; AUG) of the bacterial mRNA target sequence. For example, inparticular embodiments, the 5′-end of the target sequence is theadenine, uracil, or guanine nucleotide in a translational start codon ofthe bacterial mRNA. In some embodiments, the 5′-end or 3′-end of thetarget sequence begins at residue 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 downstream of the last nucleotide (e.g., guanine) of a translationalstart codon of the bacterial mRNA. In some embodiments, the 5′-end or3′-end of the target sequence begins at residue 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 upstream of the first nucleotide (e.g., adenine) of atranslational start codon of the bacterial mRNA

In some embodiments, the virulence factor is an antibiotic resistancegene or its encoded protein, i.e., a gene or protein that is associatedwith resistance of the bacteria to at least one antimicrobial agent.General examples of antibiotic resistance genes include beta-lactamases,which can enzymatically deactivate certain antimicrobial agents, andproteins that increase the permeability or active efflux (pumping-out)of an antimicrobial agent. Particular examples of antibiotic resistancegenes include New Delhi metallo-beta-lactamase (NDM-1) andresistance-nodulation-cell division (RND)-type multidrug efflux pumpsubunit AdeA (adeA). Exemplary translational start codon regionsequences of the NDM-1 and AdeA resistance genes are provided in Table1A below.

In some embodiments, the virulence factor is a biofilm formation gene orits encoded protein, i.e., a gene or protein that is associated with orcontributes to the formation of biofilm. A biofilm can include any groupof bacterial cells that adhere to each other on a surface, for example,a tissue surface or a surface of an implanted medical device. Suchadherent cells are often embedded within a self-produced matrix ofextracellular polymeric substance (EPS), a polymeric mixture composed,for example, of extracellular DNA, proteins, and polysaccharides.Bacteria form a biofilm in response to many factors, which may includecellular recognition of specific or non-specific attachment sites on asurface, nutritional cues, or in some cases, by exposure of cells tosub-inhibitory concentrations of antibiotics. The microbial cellsgrowing in a biofilm are physiologically distinct from individual cellsof the same organism. For example, when a bacterial cell switches to thebiofilm mode of growth, it undergoes a phenotypic shift in behavior inwhich certain genes (e.g., biofilm formation-associated) aredifferentially regulated. Particular examples of biofilm formation genesinclude cepI, cepR, and suhB. In particular embodiments, the cepI geneis from a Burkholderia species or sub-species (e.g., Burkholderiacepacia complex, Burkholderia cenocepacia) and encodes an acylhomoserinelactone synthase. In some embodiments, the suhB gene is from aBurkholderia species or sub-species (e.g., Burkholderia cepacia complex,Burkholderia cenocepacia) and encodes a putativeinositol-1-monophosphatase. In certain embodiments, the cepR gene isfrom a Burkholderia species or sub-species (e.g., Burkholderia cepaciacomplex, Burkholderia cenocepacia) and encodes an acylhomoserine lactonedependent regulatory protein. Exemplary translational start codon regionsequences of biofilm formation genes from Burkholderia are provided inTable 1B below.

In some embodiments, the virulence factor is a gene or protein that isassociated with biosynthesis of fatty acids. General examples ofproteins associated with fatty acid biosynthesis include: acyl carrierprotein (ACP), such as AcpP, that plays an essential role in stabilizingand shuttling the intermediate fatty acid chain to each of the enzymesin the fatty acid synthase complex; acyl carrier protein synthase(AcpS), an enzyme that transfers the 4′-phosphopantetheine prostheticgroup to apo-ACP to form the functional holo-ACP; acetyl-CoAcarboxylase, an enzyme composed of four proteins that catalyzes theconversion of acetyl-CoA to malonyl-CoA in the first committed step offatty acid biosynthesis; fatty acid biosynthesis (Fab) enzymes, such asFabA, FabI, FabF, FabB, FabD, FabH, FabG and FabZ, that each catalyzeeither elongation or tailoring steps on the growing fatty acid chain. Aparticular example of a gene associated with fatty acid biosynthesisincludes the acyl carrier protein acpP gene. An exemplary translationalstart codon region sequence of the acyl carrier protein acpP gene isprovided in Table 1C below.

TABLE 1A Exemplary Antibiotic Resistance Target Sequences SEQ IDDescription Sequence* NO: E. coli New GTTTTTAATG CTGAATAAAA  1Delhi Metallo- GGAAAACTTG ATGGAATTGC  beta- CCAATATTAT GCACCCGGTClactamase- 1 (NDM-1) Klebsiella GTTTTTAATG CTGAATAAAA  2 pneumoniaeGGAAAACTTG ATGGAATTGC  clone KPM_ CCAATATTAT GCACCCGGTC nasey New Delhimetallo-beta- lactamase 1 (blaNDM-1)  gene AcinetobacterAACATCAAAA AGTCACTAGG  3 baumannii TTTGGACAGT ATGCAAAAGC  metallo-beta-ATCTTTTACT TCCTTTATTT lactamase Acinetobacter AACATCAAAA AGTCACTAGG  4baumannii 1605 TTTGGACAGT ATGCAAAAGC  RND-type ATCTTTTACT TCCTTTATTTmultidrug efflux pump subunit AdeA

TABLE 1B Exemplary Biofilm Formation Target Sequences SEQ ID DescriptionSequence* NO: cepI GCATACAAAA GCACAGATCC GAGGACATCC ATGCAGACCT 5Burkholderia TCGTTCACGA GGAAGGGCGG cenocepacia J2315 N- acylhomoserinelactone synthase cepI TCACTTGAAA AATAAGTGGA AGCACTTGTA ATGAATATTA 6Actinetobacter TTGCTGGATT TCAAAACAAT baumannii AB307-0294 suhBTCTTCAAATT TGTATTGTAG TGGGTGTTCA ATGGAACCTA 7 ActinetobacterTGGTGGTGAT GGCTGCGCGT baumannii AYE SuhBCCCGTGCCGC CGGCTACAGG ATCCAGGCTC ATGCATCCCA 8 BurkholderiaTGCTCAACAT TGCTGTCAAG cenocepacia J2315 Inositol- 1-monophosphatesuhB Gene ID: CCCGTGCCGCCGGCTACAGGATCCAGGCTCATGCATCCCATGCTCAACATTG 96932290 Locus CTGTCAAGGCTGCGCGCCGCGCCGGACAGATCATCAATCGCGCGTCCCTCGA Tag BCAL2157 TCTCGACCTGATCGAGATCCGCAAGAAGCAGCAGAACGACTTCGTCACCGAAGTGGACAAGGCCGCCGAAGACGCGATCATCGAGACGCTGAAGACCGCCTACCCCGACCACGCGATCCTCGCGGAGGAATCGGGCGAATCCGACAACGAATCCGAATTCAAGTGGATCATCGATCCGCTCGACGGCACGACCAACTTCATCCACGGCTTCCCGTATTACTGCGTATCGATCGCGCTCGAGCACAAGGGCGTCGTCACGCAGGCCGTCGTCTACGATCCGAACAAGAACGACCTGTTCACGGCCACCCGCGGCCGCGGCGCATACCTGAACGACCGCCGCATCCGCGTCGGCCGCCGCGACCGCCTGGCAGACGCACTGGTCGGCACGGGCTTCCCGTTCCGCGAGAAGGACGGCCTCGACGCCTACGCGCGCCTCTTCACCGAAATGACGCAGGCCTGCACGGGCCTGCGCCGTCCGGGCGCGGCGGCGCTCGATCTCGCGAACGTCGCGGCCGGCCGCCTCGACGCGTTCTTCGAGCAAGGCATCAACGTGTGGGACATGGCAGCGGGCAGCCTGCTGATCACCGAGGCCGGCGGCCTCGTCGGGAACTACACGGGCGACGCCGATTTCCTGCATCGCCACGAGATCGTCGCCGCGAACCC

TABLE 1C Exemplary Fatty Acid Biosynthesis-Associated Target SequenceSEQ ID Description Sequence* NO: acpP GCGCACTTGTAAATCTGAACTTTCCCTCGGA 10acyl carrier GGGGTAATGACAACATCGAACAACGTGTCAA protein GCCGATGTCGCTGAACAA*The thymines (T) can be uracils (0)

Thus, in certain embodiments, antisense targeting sequences are designedto hybridize to a region of one or more of the target sequences listedin Table 1 or a target gene described herein. Selected antisensetargeting sequences can be made shorter, e.g., about 8, 9, 10, 11, 12,13, 14, or 15 bases, or longer, e.g., about 20, 30, or 40 bases, andinclude a small number of mismatches, as long as the sequence issufficiently complementary to reduce transcription or translation uponhybridization to the target sequence, and optionally forms with the RNAa heteroduplex having a Tm of 45° C. or greater.

In certain embodiments, the degree of complementarity between the targetsequence and antisense targeting sequence is sufficient to form a stableduplex. The region of complementarity of the antisense oligomers withthe target RNA sequence may be as short as 8-9 bases, 8-10 bases, 8-11bases, 8-12 bases, 10-11 bases, 10-12 bases, but can be 12-15 bases ormore, e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20bases, or 15-20 bases, including all integers in between these ranges.An antisense oligomer of about 10-15 bases is generally long enough tohave a unique complementary sequence. In certain embodiments, a minimumlength of complementary bases may be required to achieve the requisitebinding Tm, as discussed herein.

In certain embodiments, oligomers as long as 40 bases may be suitable,where at least a minimum number of bases, e.g., 10-12 bases, arecomplementary to the target sequence. In general, however, facilitatedor active uptake in cells is optimized at oligomer lengths of less thanabout 30 or less than about 20 bases. Included are antisense oligomersthat consist of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, or 40 bases, in which at least about 6, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 contiguous or non-contiguous bases arecomplementary to a target gene described herein, for example, a targetsequence of Table 1 (e.g., SEQ ID NOS: 1-10).

In certain embodiments, antisense oligomers may be 100% complementary tothe target sequence, or may include mismatches, e.g., to accommodatevariants, as long as a heteroduplex formed between the oligomer andtarget sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo, and reduce expression of the targeted mRNA. Hence, certainoligomers may have about or at least about 70% sequence complementarity,e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence complementarity, between the oligomer andthe target sequence. Oligomer backbones that are less susceptible tocleavage by nucleases are discussed herein. Mismatches, if present, aretypically less destabilizing toward the end regions of the hybrid duplexthan in the middle. The number of mismatches allowed will depend on thelength of the oligomer, the percentage of G:C base pairs in the duplex,and the position of the mismatch(es) in the duplex, according to wellunderstood principles of duplex stability. Although such an antisenseoligomer is not necessarily 100% complementary to the target sequence,it is effective to stably and specifically bind to the target sequence,for example, such that translation of the target RNA is reduced.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an oligomer withrespect to complementary-sequence RNA may be measured by conventionalmethods, such as those described by Hames et al., Nucleic AcidHybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C.G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, MethodsEnzymol. Vol. 154 pp. 94-107. In certain embodiments, antisenseoligomers may have a binding Tm, with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than about 45° C. or 50° C. Tm's in the range 60-80°C. or greater are also included. According to well-known principles, theTm of an oligomer, with respect to a complementary-based RNA hybrid, canbe increased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer.

Tables 2A-C below shows exemplary targeting sequences (in a 5′-to-3′orientation) of antisense oligomers described herein.

TABLE 2A Exemplary Antibiotic Resistance Targeting Sequences TargetTS SEQ Gene Targeting Sequence (TS)* ID NO: NDM-1 TCA AGT TTT CC 11NDM-1 TCC TTT TAT TC 12 NDM-1 CCA TCA AGT TT 13 NDM-1 GGC AAT TCC AT 14adeA ATA CTG TCC AA 15

TABLE 2B Exemplary Biofilm Formation Targeting Sequences Target TS SEQGene Targeting Sequence (TS)* ID NO: cepI AAG GTC TGC AT 16 cepITCG GAT CTG TG 17 cepI CAT GGA TGT CC 18 cepI CGT GAA CGA AG 19 cepICGT GTG GCA AC 20 cepI GCC CGA GAT CC 21 cepI CTT TCG TTC GC 22 suhBATG CAT GAG CC 23 suhB GGA TGC ATG AG 24

TABLE 2C Exemplary Fatty Acid Biosynthesis-Associated Targeting Sequences Target TS SEQ GeneTargeting Sequence (TS)* ID NO: acpP GTCCATTACCC 25 acpP CATTACCCCTC 26acpP CCATTACCCCT 27 acpP TCCATTACCCC 28 acpP TGTCCATTACC 29 acpPTTGTCCATTAC 30 acpP GTTGTCCATTA 31 acpP TGTTGTCCATT 32 acpP ATGTTGTCCAT33 acpP TTTACAAGTGC 34 acpP CCTCCGAGGGA 35 acpP ACACGTTGTTC 36 acpPAGTTCAGCGAC 37

*The thymines (T) can be uracils (U).

Certain antisense oligomers thus comprise, consist, or consistessentially of a targeting sequence in Table 2 (e.g., SEQ ID NOS: 11-37)or a variant or contiguous or non-contiguous portion(s) thereof. Forinstance, certain antisense oligomers comprise about or at least about6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, or 27 contiguous or non-contiguous nucleotides of any of thetargeting sequences in Table 2 (e.g., SEQ ID NOS: 11-37). Fornon-contiguous portions, intervening nucleotides can be deleted orsubstituted with a different nucleotide, or intervening nucleotides canbe added. Additional examples of variants include oligomers having aboutor at least about 70% sequence identity or homology, e.g., 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity or homology, over the entire length of any of thetargeting sequences in Table 2 (e.g., SEQ ID NOS: 11-37).

The activity of antisense oligomers and variants thereof can be assayedaccording to routine techniques in the art (see, e.g., the Examples).

I. Antisense Oligomer Compounds

The antisense oligomers typically comprises a base sequence ofsufficient length and complementarity to specifically hybridize to abacterial mRNA target sequence that encodes a virulence factor, andthereby reduce expression (e.g., translation) of the virulence factorprotein. This requirement is optionally met when the oligomer compoundhas the ability to be actively taken up by bacterial cells, and oncetaken up, form a stable duplex (or heteroduplex) with the target mRNA,optionally with a Tm greater than about 40° C. or 45° C.

A. Antisense Oligomer Chemical Features

In certain embodiments, the backbone of the antisense oligomer issubstantially uncharged, and is optionally recognized as a substrate foractive or facilitated transport across a cell wall and/or cell membrane.The ability of the oligomer to form a stable duplex with the target RNAmay also relate to other features of the backbone, including the lengthand degree of complementarity of the antisense oligomer with respect tothe target, the ratio of G:C to A:T base matches, and the positions ofany mismatched bases. The ability of the antisense oligomer to resistcellular nucleases may promote survival and ultimate delivery of theagent to the cell. Exemplary antisense oligomer targeting sequences arelisted in Table 2 (supra).

In certain embodiments, the antisense oligomer is a morpholino-basedoligomer, for example, a phosphorodiamidate morpholino oligomer (PMO).Morpholino-based oligomers refer to an oligomer comprising morpholinosubunits supporting a nucleobase and, instead of a ribose, contains amorpholine ring. Exemplary internucleoside linkages include, forexample, phosphoramidate or phosphorodiamidate internucleoside linkagesjoining the morpholine ring nitrogen of one morpholino subunit to the 4′exocyclic carbon of an adjacent morpholino subunit. Each morpholinosubunit comprises a purine or pyrimidine nucleobase effective to bind,by base-specific hydrogen bonding, to a base in an oligonucleotide.

Morpholino-based oligomers (including antisense oligomers) are detailed,for example, in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047;5,034,506; 5,166,315; 5,185,444; 5,521,063; 5,506,337 and pending U.S.patent application Ser. Nos. 12/271,036; 12/271,040; and PCT PublicationNo. WO/2009/064471 and WO/2012/043730 and Summerton et al. 1997,Antisense and Nucleic Acid Drug Development, 7, 187-195, which arehereby incorporated by reference in their entirety.

Within the oligomer structure, the phosphate groups are commonlyreferred to as forming the “internucleoside linkages” of the oligomer.The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. A “phosphoramidate” group comprisesphosphorus having three attached oxygen atoms and one attached nitrogenatom, while a “phosphorodiamidate” group comprises phosphorus having twoattached oxygen atoms and two attached nitrogen atoms. In the unchargedor the cationic internucleoside linkages of the morpholino-basedoligomers described herein, one nitrogen is always pendant to thelinkage chain. The second nitrogen, in a phosphorodiamidate linkage, istypically the ring nitrogen in a morpholine ring structure.

Accordingly, various embodiments of the disclosure include asubstantially uncharged antisense morpholino oligomer, composed ofmorpholino subunits and phosphorus-containing intersubunit linkagesjoining a morpholino nitrogen of one subunit to a 5′-exocyclic carbon ofan adjacent subunit, and having (a) about 10-40 nucleotide bases, and(b) a targeting sequence of sufficient length and complementarity tospecifically hybridize to a bacterial mRNA target sequence that encodesa virulence factor; where the oligomer is conjugated to acell-penetrating peptide (CPP). In particular embodiments, themorpholino subunits are joined by phosphorous-containing intersubunitlinkages in accordance with the structure:

where Y₁=oxygen (O) or sulfur, nitrogen, or carbon; Z=oxygen or sulfur,preferably oxygen; Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is —NRR′ where R and R′ are the same or differentand are either H or alkyl. In particular embodiments, X is —NRR′, whereR and R′ are the same or different and are either H or methyl.

Also included are antisense oligomer that comprise a sequence ofnucleotides of the formula in FIGS. 1A-1E. In FIG. 1A, B is a purine orpyrimidine base-pairing moiety effective to bind, by base-specifichydrogen bonding, to a base in a polynucleotide. Y₁ or Y₂ may be oxygen,sulfur, nitrogen, or carbon, preferably oxygen. The X moiety pendantfrom the phosphorus may be fluorine, an alkyl or substituted alkyl, analkoxy or substituted alkoxy, a thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures, such as morpholines or piperidines. Alkyl, alkoxy andthioalkoxy include 1-6 carbon atoms. The Z moieties may be sulfur oroxygen, and are preferably oxygen.

In various aspects, an antisense oligomer of the disclosure includes acompound of formula (I):

or a pharmaceutically acceptable salt thereof,

where each Nu is a nucleobase which taken together forms a targetingsequence;

X is an integer from 9 to 38;

T is selected from OH and a moiety of the formula:

where each R⁴ is independently C₁-C₆ alkyl, and R⁵ is selected from anelectron pair and H, and R⁶ is selected from OH, —N(R⁷)CH₂C(O)NH₂, and amoiety of the formula:

where:

-   -   R⁷ is selected from H and C₁-C₆ alkyl, and    -   R⁸ is selected from G, —C(O)—R⁹OH, acyl, trityl, and        4-methoxytrityl, where:        -   R⁹ is of the formula —(O-alkyl)_(y)- wherein y is an integer            from 3 to 10 and each of the y alkyl groups is independently            selected from C₂-C₆ alkyl;    -   each instance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is        independently C₁-C₆ alkyl, and R¹¹ is selected from an electron        pair and H;    -   R² is selected from H, G, acyl, trityl, 4-methoxytrityl,        benzoyl, stearoyl, and a moiety of the formula:

where L is selected from —C(O)(CH₂)₆C(O)— and —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,and each R¹² is of the formula —(CH₂)₂OC(O)N(R¹⁴)₂ wherein each R¹⁴ isof the formula —(CH₂)₆NHC(═NH)NH₂; and

R³ is selected from an electron pair, H, and C₁-C₆ alkyl,

wherein G is a cell penetrating peptide (“CPP”) and linker moietyselected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,—C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP,

and —C(O)CH₂NH—CPP, or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus, with the proviso that only one instance of G ispresent,

wherein the targeting sequence specifically hybridizes to a bacterialmRNA target sequence that encodes a virulence factor.

In some embodiments, X is from 9 to 18. In certain embodiments, X is 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30.

In certain embodiments, T is selected from:

In some embodiments, R² is selected from H, G, acyl, trityl,4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, T is selected from:

and R² is G.

In some embodiments, T is of the formula:

R⁶ is of the formula:

and R² is G.

In certain embodiments, T is of the formula:

and R² is G.

In certain embodiments, T is of the formula:

In some embodiments, R² is G or T is of the formula:

In some embodiments, R² is selected from H, acyl, trityl,4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, R² is selected from H or G, and R³ is selectedfrom an electron pair or H. In a particular embodiment, R² is G. In someembodiments, R² is H or acyl. In some embodiments, each R¹ is —N(CH₃)₂.In some embodiments, at least one instance of R¹ is —N(CH₃)₂. In certainembodiments, each instance of R¹ is —N(CH₃)₂.

In various embodiments of the disclosure, an antisense oligomer of thedisclosure includes a compound of formula (II):

or a pharmaceutically acceptable salt thereof,

where each Nu is a nucleobase which taken together forms a targetingsequence;

X is an integer from 9 to 28;

T is selected from:

R² is selected from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, andstearoyl; and

R³ is selected from an electron pair, H, and C₁-C₆ alkyl,

wherein G is a cell penetrating peptide (“CPP”) and linker moietyselected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,—C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, and —C(O)CH₂NH—CPP, or G is of theformula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus, with the proviso that only one instance of G ispresent. In various embodiments, R² is G or T is of the formula:

In some embodiments, T is TEG as defined above, R² is G, and R³ is anelectron pair or H. In certain embodiments, R² is selected from H, acyl,trityl, 4-methoxytrityl, benzoyl, and stearoyl and T is of the formula:

In various aspects, an antisense oligomer of the disclosure includes acompound of formula (III):

or a pharmaceutically acceptable salt thereof,

where each Nu is a nucleobase which taken together forms a targetingsequence;

X is an integer from 9 to 28;

T is selected from:

each instance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is independentlyC₁-C₆ alkyl, and R¹¹ is selected from an electron pair and H;

R² is selected from an electron pair, H, and C₁-C₆ alkyl; and

G is a cell penetrating peptide (“CPP”) and linker moiety selected from—C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP, —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP,

and —C(O)CH₂NH—CPP, or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus.In some embodiments, at least one instance of R¹ is —N(CH₃)₂. In certainembodiments, each instance of R¹ is —N(CH₃)₂.

In various aspects, an antisense oligomer of the disclosure includes acompound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

X is an integer from 9 to 28;

each Nu is a nucleobase which taken together forms a targeting sequence;

each instance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is independentlyC₁-C₆ alkyl, and R¹¹ is selected from an electron pair and H; and

G is a cell penetrating peptide (“CPP”) and linker moiety selected from—C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP, —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP,

and —C(O)CH₂NH—CPP, or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus. In some embodiments, at least one instance of R¹is —N(CH₃)₂. In certain embodiments, each instance of R¹ is —N(CH₃)₂.

In various aspects, an antisense oligomer of the disclosure can be acompound of formula (V):

wherein:

X is an integer from 9 to 18;

each Nu is a nucleobase which taken together forms a targeting sequence;

each instance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is independentlyC₁-C₆ alkyl, and R¹¹ is selected from an electron pair and H;

R² is selected from H, trityl, 4-methoxytrityl, acyl, benzoyl, andstearoyl; and

R³ is selected from an electron pair, H, and C₁-C₆ alkyl,

wherein G is a cell penetrating peptide (“CPP”) and linker moietyselected from —C(O)(CH₂)₃NH—CPP, —C(O)(CH₂)₂NH—CPP,—C(O)(CH₂)₂NHC(O)(CH₂)₃NH—CPP,

and —C(O)CH₂NH—CPP, or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus. In some embodiments, at least one instance of R¹is —N(CH₃)₂. In certain embodiments, each instance of R¹ is —N(CH₃)₂.

In various aspects, an antisense oligomer of the disclosure includes acompound of formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

X is an integer from 9 to 28;

each Nu is a nucleobase which taken together forms a targeting sequence;

R² is selected from H or acyl; and

G is a cell penetrating peptide (“CPP”) and linker moiety selected from—C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP, —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP,

and —C(O)CH₂NH—CPP, or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus.

The antisense oligomers can be prepared by stepwise solid-phasesynthesis, employing methods known in the art and described in thereferences cited herein.

B. Cell-Penetrating Peptides

In certain embodiments, the antisense oligomer is conjugated to acell-penetrating peptide (CPP). In some embodiments, the CPP is anarginine-rich peptide. By “arginine-rich carrier peptide” is meant thatthe CPP has at least 2, and preferably 2, 3, 4, 5, 6, 7, or 8 arginineresidues, each optionally separated by one or more uncharged,hydrophobic residues, and optionally containing about 6-14 amino acidresidues. FIGS. 1F-1H show exemplary chemical structures of CPP-PMOconjugates used in the Examples, including 5′ and 3′ PMO conjugates.

Exemplary CPPs are provided in Table C1 (SEQ ID NOS: 38-42).

TABLE C1 Exemplary Cell-Penetrating Peptides Name Sequence SEQ ID NO:(RXR)₄ RXRRXRRXRRXR 38 (RFF)₃R RFFRFFRFFR 39 (RXR)₄XB RXRRXRRXRRXRXB 40(RFF)₃RXB RFFRFFRFFRXB 41 (RFF)₃RG RFFRFFRFFR 42 X is 6-aminohexanoicacid; B is β-alanine; F is phenylalanine

CPPs, their synthesis, and methods of conjugating a CPP to an oligomerare detailed, for example, in International Patent ApplicationPublication Nos. WO 2004/097017, WO 2009/005793, and WO 2012/150960,which are all incorporated by reference in their entirety.

In some embodiments, the CPP is linked at its C-terminus to the 3′-endor the 5′-end of the oligomer via a 1, 2, 3, 4, or 5 amino acid linker.In particular embodiments, including antisense oligomer compounds offormula (I)-(VI), the linkers can include: —C(O)(CH₂)₅NH—CPP (X linker),—C(O)(CH₂)₂NH—CPP (B linker), —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP (XB peptidelinker), and —C(O)CH₂NH—CPP (Gly linker), or G is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus. In some embodiments of the disclosure, includingantisense oligomer compounds of formula (I)-(VI), G is selected from SEQID NOS: 40 to 42. In various embodiments, including antisense oligomercompounds of formula (I)-(VI), the CPP is selected from SEQ ID NO: 38and 39, and the linker is selected from the group described above.

In some embodiments, including antisense oligomer compounds of formula(I)-(VI), the CPP is selected from:

wherein R^(a) is selected from H, acetyl, benzoyl, and stearoyl.

In some embodiments, including antisense oligomer compounds of formula(I)-(VI), G is selected from:

wherein R^(a) is selected from H, acetyl, benzoyl, and stearoyl.

In various aspects, an antisense oligomer of the disclosure, or apharmaceutically acceptable salt thereof, includes an antisense oligomerof the formula (VII) selected from:

wherein X is an integer from 9 to 38, R^(a) is selected from H, acetyl,benzoyl, and stearoyl, R^(b) is selected from H, acetyl, benzoyl,stearoyl, trityl, and 4-methoxytrityl, and each Nu is a purine orpyrimidine base-pairing moiety which taken together form a targetingsequence described above.

C. Antisense Oligomer Targeting Sequence

In various embodiments of the antisense oligomers of the disclosure,including the antisense oligomer compounds of formulas (I)-(VII), thetargeting sequence can specifically hybridizes to a bacterial mRNAtarget sequence that encodes a virulence factor. In some embodiments,the target sequence comprises a translational start codon of thebacterial mRNA and/or a sequence within about 30 bases upstream ordownstream of the translational start codon of the bacterial mRNA. Incertain embodiments, the virulence factor can be an antibioticresistance protein or a biofilm formation protein. In some embodiments,the antibiotic resistance protein may be selected from at least one ofNew Delhi metallo-beta-lactamase (NDM-1) and resistance-nodulation-celldivision (RND)-type multidrug efflux pump subunit AdeA (adeA). In someembodiments, the target sequence can be selected from SEQ ID NOS: 1-4,wherein thymine bases (T) are optionally uracil bases (U). In certainembodiments, the targeting sequence may be one of the targetingsequences set forth in SEQ ID NOS: 11-15, may comprise a fragment of atleast 10 contiguous nucleotides of SEQ ID NOS: 11-15, or may comprise avariant having at least 80% sequence identity to SEQ ID NOS: 11-15,wherein thymine bases (T) are optionally uracil bases (U). In someembodiments, the biofilm formation protein may be encoded by at leastone of CepI or SuhB. In certain embodiments, the target sequence can beselected from SEQ ID NOS: 5-9, wherein thymine bases (T) are optionallyuracil bases (U). In some embodiments, the targeting sequence may be oneof the targeting sequences set forth in SEQ ID NOS: 16-24, may comprisea fragment of at least 10 contiguous nucleotides of SEQ ID NOS: 16-24,or may comprise a variant having at least 80% sequence identity to SEQID NOS: 16-24, wherein thymine bases (T) are optionally uracil bases(U). In various embodiments, the virulence factor is an acyl carrierprotein associated with fatty acid biosynthesis encoded by one or moreof acpP. In certain embodiments, the acyl carrier protein may be AcpP.In some embodiments, the target sequence may be SEQ ID NO: 10, whereinthymine bases (T) are optionally uracil bases (U). In certainembodiments, the targeting sequence may be one of the targetingsequences set forth in SEQ ID NOS: 25-37, may comprise a fragment of atleast 10 contiguous nucleotides of SEQ ID NOS: 25-37, or may comprise avariant having at least 80% sequence identity to SEQ ID NOS: 25-37,wherein thymine bases (T) are optionally uracil bases (U). In someembodiments of the disclosure, including the antisense oligomercompounds of formulas (I)-(VII), the targeting sequence is selectedfrom:

a) SEQ ID NO: 11 (TCA AGT TTT CC); b) SEQ ID NO: 12 (TCC TTT TAT TC);c) SEQ ID NO: 13 (CCA TCA AGT TT); d) SEQ ID NO: 14 (GGC AAT TCC AT);and e) SEQ ID NO: 15 (ATA CTG TCC AA),

wherein X is 9, and thymine bases (T) may be uracil bases (U).

In various embodiments of the disclosure, including the antisenseoligomer compounds of formulas (I)-(VII), the targeting sequence isselected from:

a) SEQ ID NO: 16 (AAG GTC TGC AT); b) SEQ ID NO: 17 (TCG GAT CTG TG);c) SEQ ID NO: 18 (CAT GGA TGT CC); d) SEQ ID NO: 19 (CGT GAA CGA AG);e) SEQ ID NO: 20 (CGT GTG GCA AC); f) SEQ ID NO: 21 (GCC CGA GAT CC);g) SEQ ID NO: 22 (CTT TCG TTC GC); h) SEQ ID NO: 23 (ATG CAT GAG CC);and i) SEQ ID NO: 24 (GGA TGC ATG AG),

wherein X is 9, and thymine bases (T) may be uracil bases (U).

In certain embodiments of the disclosure, including the antisenseoligomer compounds of formulas (I)-(VII), the targeting sequence isselected from:

a) SEQ ID NO: 25 (GTCCATTACCC); b) SEQ ID NO: 26 (CATTACCCCTC);c) SEQ ID NO: 27 (CCATTACCCCT); d) SEQ ID NO: 28 (TCCATTACCCC);e) SEQ ID NO: 29 (TGTCCATTACC); f) SEQ ID NO: 30 (TTGTCCATTAC);g) SEQ ID NO: 31 (GTTGTCCATTA); h) SEQ ID NO: 32 (TGTTGTCCATT);i) SEQ ID NO: 33 (ATGTTGTCCAT); j) SEQ ID NO: 34 (TTTACAAGTGC);k) SEQ ID NO: 35 (CCTCCGAGGGA); l) SEQ ID NO: 36 (ACACGTTGTTC);m) SEQ ID NO: 37 (AGTTCAGCGAC),

wherein X is 9, and thymine bases (T) may be uracil bases (U).

D. Exemplary Antisense Oligomers

Exemplary antisense oligomers (AONs) of the disclosure include thosedescribed in Tables 3A-3C below.

TABLE 3A Exemplary Antibiotic Resistance Targeting Sequences AONs PMOTarget Targeting Sequence TS SEQ 5′ 3′ Attachment CPP SEQ Name Gene(TS)* ID NO: Attachment ** ID NO. PPMO#1 NDM-1 TCA AGT TTT CC 11 TEG(RXR)₄XB- 40 PPMO#2 NDM-1 TCC TTT TAT TC 12 TEG (RXR)₄XB- 40 PPMO#3NDM-1 CCA TCA AGT TT 13 TEG (RXR)₄XB- 40 PPMO#4 NDM-1 GGC AAT TCC AT 14TEG (RXR)₄XB- 40 PPMO#5 adeA ATA CTG TCC AA 15 TEG (RXR)₄XB- 40 * Thethymines (T) can be uracils (U); ** X is 6-aminohexanoic acid, B isbeta-alanine, G is glycine, F is phenylalanine, and TEG is definedabove.

TABLE 3B Exemplary Biofilm Formation Targeting AONs CPP PMO TargetTargeting Sequence TS SEQ 5′ Attachment 3′ Attachment SEQ ID Name Gene(TS)* ID NO: *** ** NO. PPMO#6 cepI AAG GTC TGC AT 16 (RFF)₃RXB- H 41PPMO#7 cepI TCG GAT CTG TG 17 TEG (RFF)₃RXB- 41 PPMO#8 cepICAT GGA TGT CC 18 TEG (RFF)₃RXB- 41 PPMO#9 cepI CGT GAA CGA AG 19 TEG(RFF)₃RXB- 41 PPMO#10 cepI CGT GTG GCA AC 20 TEG (RFF)₃RXB- 41 PPMO#11cepI GCC CGA GAT CC 21 TEG (RFF)₃RXB- 41 PPMO#12 cepI CTT TCG TTC GC 22TEG (RFF)₃RXB- 41 PPMO#13 suhB ATG CAT GAG CC 23 TEG (RFF)₃RXB- 41PPMO#14 suhB GGA TGC ATG AG 24 TEG (RFF)₃RXB- 41 * The thymines (T) canbe uracils (U); ** X is 6-aminohexanoic acid, B is beta-alanine, G isglycine, F is phenylalanine, and TEG is defined above. *** X is6-aminohexanoic acid, B is beta-alanine, G is glycine, F isphenylalanine, and a 5′ CPP is linked through a pip-PDA moiety describedabove.

TABLE 3CExemplary Fatty Acid Biosynthesis-Associated Targeting Sequences AONsTargeting CPP PMO Target Sequence TS SEQ 5′ Attachment 3′ AttachmentSEQ ID Name Gene (TS)* ID NO: *** ** NO. PPMO#15 acpP GTCCATTACCC 25(RFF)₃RXB- H 41 PPMO#16 acpP GTCCATTACCC 25 TEG (RFF)₃RXB- 41 PPMO#17acpP GTCCATTACCC 25 (RFF)₃RG- H 42 PPMO#18 acpP CATTACCCCTC 26(RFF)₃RXB- H 41 PPMO#19 acpP CCATTACCCCT 27 (RFF)₃RXB- H 41 PPMO#20 acpPTCCATTACCCC 28 (RFF)₃RXB- H 41 PPMO#21 acpP TGTCCATTACC 29 (RFF)₃RXB- H41 PPMO#22 acpP TTGTCCATTAC 30 (RFF)₃RXB- H 41 PPMO#23 acpP GTTGTCCATTA31 (RFF)₃RXB- H 41 PPMO#24 acpP TGTTGTCCATT 32 (RFF)₃RXB- H 41 PPMO#25acpP ATGTTGTCCAT 33 (RFF)₃RXB- H 41 PPMO#26 acpP TTTACAAGTGC 34 TEG(RFF)₃RXB- 41 PPMO#27 acpP CCTCCGAGGGA 35 TEG (RFF)₃RXB- 41 PPMO#28 acpPACACGTTGTTC 36 TEG (RFF)₃RXB- 41 PPMO#29 acpP AGTTCAGCGAC 37 TEG(RFF)₃RXB- 41 * The thymines (T) can be uracils (U); ** X is6-aminohexanoicacid, B is beta-alanine, G is glycine, F isphenylalanine, and TEG is defined above. *** X is 6-aminohexanoic acid,B is beta-alanine, G is glycine, F is phenylalanine, and a 5′ CPP islinked through a pip-PDA moiety described above.

II. Methods of Use and Formulations

Embodiments of the present disclosure include methods of using theantisense oligomers described herein to reduce the expression andactivity of one or more bacterial virulence factors. Certain embodimentsinclude methods of using the antisense oligomers to reduce replication,proliferation, virulence factors, or growth of bacteria, for example, totreat bacterial infections in a subject, either alone or in combinationwith one or more additional antimicrobial agents. In some instances, theantisense oligomers increase the susceptibility of the bacterium toantibiotics. Certain embodiments include methods of using the antisenseoligomers described herein to reduce the formation or existence ofbacterial biofilms, for instance, to treat bacterial infections in asubject, either alone or in combination with one or more additionalantimicrobial agents.

Also included are pharmaceutical compositions comprising the antisenseoligomers, typically in combination with a pharmaceutically-acceptablecarrier. The methods provided herein can be practiced in vitro or invivo.

For example, certain embodiments include methods of treating a bacterialinfection in a subject, comprising administering to a subject in needthereof (e.g., subject having or at risk for having a bacterialinfection) an antisense oligomer or pharmaceutical composition describedherein. Also included are methods of reducing virulence and/or biofilmformation of a bacteria or bacterium which comprises a gene encoding avirulence factor, comprising contacting the bacteria or bacterium withan antisense oligomer described herein.

In some embodiments, the bacterium is selected from the genusEscherichia, Acinetobacter, Klebsiella, Burkholderia, and Pseudomonas.

Escherichia is a genus of Gram-negative, non-spore forming,facultatively anaerobic, rod-shaped bacteria from the familyEnterobacteriaceae, and includes the species Escherichia coli, which isresponsible for the vast majority of Escherichia-related pathogenesis.

Acinetobacter is a genus of Gram-negative bacteria belonging to theclass of Gammaproteobacteria. Examples of clinically-relevantAcinetobacter complexes include the Acinetobacter calcoaceticus-baumaniicomplex (glucose-oxidizing nonhemolytic), Acinetobacter lwoffii(glucose-negative nonhemolytic), and Acinetobacter haemolyticus(hemolytic). Specific examples include Acinetobacter baumannii.

Klebsiella is a genus of non-motile, Gram-negative, oxidase-negative,rod-shaped bacteria with a prominent polysaccharide-based capsule.Klebsiella organisms can lead to a wide range of disease states, such aspneumonia, urinary tract infections, septicemia, meningitis, diarrhea,and soft tissue infections. The majority of human infections are causedby Klebsiella pneumoniae and Klebsiella oxytoca.

Burkholderia (previously part of Pseudomonas) refers to a group of nearubiquitous gram-negative, motile, obligately aerobic rod-shapedbacteria. These protobacteria include pathogenic bacteria such asBurkholderia mallei, responsible for glanders; Burkholderiapseudomallei, causative agent of melioidosis; and Burkholderia cepacia,a significant pathogen of pulmonary infections, for example, in subjectswith cystic fibrosis (CF). Burkholderia cepacia (or Burkholderia cepaciacomplex) is a Gram-negative bacterium composed of many differentsub-species, including, for example, Burkholderia cenocepacia,Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderiastabilis, Burkholderia anthina, Burkholderia pyrrocinia, Burkholderiadolosa, and/or Burkholderia ambifaria.

Pseudomonas is a genus of Gram-negative aerobic gammaproteobacteria,belonging to the family Pseudomonadaceae. Pseudomonas aeruginosa isincreasingly recognized as an emerging opportunistic pathogen ofclinical relevance. It has low antibiotic susceptibility and can formbiofilms. Pseudomonas spp. are naturally resistant to penicillin and themajority of related beta-lactam antibiotics, but some are sensitive topiperacillin, imipenem, ticarcillin, and/or ciprofloxacin.Aminoglycosides such as tobramycin, gentamicin, and amikacin are otherpotential microbial agents for the treatment of Pseudomonas infections.

Thus, in some embodiments, the bacterium is any of the foregoing membersof the genera Escherichia, Acinetobacter, Klebsiella, Burkholderia, andPseudomonas. In specific embodiments, the bacterium is one or more ofEscherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae,Burkholderia cepacia (complex), or Pseudomonas aeruginosa.

In certain embodiments, the bacterium is multi-drug resistance (MDR)bacteria or bacterium. Multiple drug resistance (MDR), multi-drugresistance or multiresistance is a condition enabling disease-causingmicroorganisms (bacteria, viruses, fungi or parasites) to resistdistinct antimicrobials such as antibiotics, antifungal drugs, antiviralmedications, antiparasitic drugs, and others. In particular embodiments,the bacterium is extensively-drug resistant (XDR) or pan-drug resistant(PDR). In some embodiments, the bacterium is an extended-spectrumβ-lactamase (ESBLs) producing Gram-negative bacteria, Klebsiellapneumoniae carbapenemase (KPC) producing Gram-negative bacteria, or amulti-drug-resistant gram negative rod (MDR GNR) MDRGN bacteria. Inspecific embodiments, the bacterium is MDR Escherichia coli, MDRAcinetobacter baumannii, MDR Klebsiella pneumoniae, MDR Burkholderiacepacia (complex), or MDR Pseudomonas aeruginosa.

As noted above, the bacteria or bacterium described herein typicallycomprise (e.g., encode) one or more virulence factors such as antibioticresistance genes, biofilm formation genes and/or genes associated withfatty acid biosynthesis. General examples of antibiotic resistance genes(and their related proteins) include beta-lactamases, which canenzymatically deactivate certain antimicrobial agents, andgenes/proteins which increase the permeability or active efflux (pumpingout) of an antimicrobial agent. Particular examples of antibioticresistance genes include New Delhi metallo-beta-lactamase (NDM-1) andresistance-nodulation-cell division (RND)-type multidrug efflux pumpsubunit AdeA (adeA). In specific embodiments, the bacterium isEscherichia coli, Acinetobacter baumannii, or Klebsiella pneumoniae,which comprises or expresses at least one antibiotic resistance geneselected from NDM-1 and adeA.

Examples of biofilm formation genes (and their related proteins) includecepI, cepR, and/or suhB genes, for example, from Burkholderia. Inparticular embodiments, the bacterium comprises or expresses the cepIgene, which encodes an acylhomoserine lactone synthase. In someembodiments, the bacterium comprises or expresses the suhB gene, whichencodes an inositol-1-monophosphate. In specific embodiments, thebacterium that comprises or expresses one more biofilm formation genesis a Burkholderia species, for example, Burkholderia cepacia orBurkholderia cepacia (complex). In some of these and relatedembodiments, the subject in need thereof is immunocompromised and has anunderlying lung disease, such as cystic fibrosis (CF) or chronicgranulomatous disease (CGD).

Examples of genes associated with fatty acid biosynthesis (and theirrelated proteins) include acpP, acpS, and/or fob genes, for example,from Burkholderia. In particular embodiments, the bacterium comprises orexpresses the acpP gene, which encodes an acyl carrier protein. Inspecific embodiments, the bacterium that comprises or expresses one ormore genes associated with fatty acid biosynthesis is a Burkholderiaspecies, for example, Burkholderia cepacia or Burkholderia cepacia(complex). In some of these and related embodiments, the subject in needthereof is immunocompromised and has an underlying lung disease, such ascystic fibrosis (CF) or chronic granulomatous disease (CGD).

In some embodiments, the antisense oligomer reduces or inhibits thegrowth of the bacterium. For instance, in some embodiments, theantisense oligomer reduces growth of the bacterium by about or at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,or 1000% or more (including all integers and ranges in between),relative to a control (e.g., absence of the antisense oligomer,scrambled oligomer, prior to contacting with the oligomer), or by aboutor at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold or more(including all integers and ranges in between), relative to a control.Bacterial growth can be measured in vitro (see, e.g., the Examples) orin vivo. In some embodiments, as described herein, the antisenseoligomer is employed in combination with one or more antimicrobialagents.

In some embodiments, the antisense oligomer reduces beta-lactamase(e.g., carbapenemase) activity in the periplasm of the bacterium byabout or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, or 1000% or more (including all integers and rangesin between), relative to a control, or by at least about 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100-fold or more (including all integers and ranges inbetween), relative to a control. In some embodiments, the antisenseoligomer reduces meropenemase enzymatic activity in the periplasm of thebacterium. In particular embodiments, the antisense oligomer thatreduces beta-lactamase (e.g., carbapenemase) activity is targetedagainst NDM-1, and the bacterium is an Acinetobacter, Escherichia, orKlebsiella species, for example, Escherichia coli, Acinetobacterbaumannii, or Klebsiella pneumoniae which comprises or expresses NDM-1.These are exemplary bacterial species and it is expected that anybacterium expressing the NDM-1 gene is susceptible to the compounds andmethods described herein. Beta-lactamase (e.g., carbapenemase) activitycan be measured according to routine techniques in the art.

In some embodiments, the antisense oligomer reduces biofilm formationand/or the levels of existing biofilm relative to a control (e.g.,absence of the oligomer). For instance, in some embodiments, theantisense oligomer reduces biofilm formation and/or the levels ofexisting biofilm by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, or 1000% or more (including allintegers and ranges in between), relative to a control, or by at leastabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100-fold or more (including allintegers and ranges in between), relative to a control. In particularembodiments, the antisense oligomer that reduces biofilm formationand/or the levels of existing biofilm is targeted against cepI, cepR,suhB, and/or acpP, and the bacterium is a Burkholderia species, forexample, Burkholderia cepacia (complex) or a sub-species thereof (e.g.,Burkholderia cenocepacia, Burkholderia multivorans, Burkholderiavietnamiensis, Burkholderia stabilis, Burkholderia anthina, Burkholderiapyrrocinia, Burkholderia dolosa, Burkholderia ambifaria), whichcomprises or expresses cepI, cepR, suhB and/or acpP. Biofilm formationand/or the levels of existing biofilm can be measured in vitro (see,e.g., the Examples) or in vivo.

In some embodiments, the methods are practiced in vivo, and compriseadministering the antisense oligomer to a subject in need thereof, forexample, a subject in need thereof that is infected or at risk for beinginfected by one or more of the bacteria or bacterium described herein.The antisense oligomers of the disclosure can thus be administered tosubjects to treat (prophylactically or therapeutically) an infection byany of the bacteria or bacterium described herein. In conjunction withsuch treatment, pharmacogenomics (e.g., the study of the relationshipbetween an individual's genotype/phenotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug.

Thus, a physician or clinician may consider applying knowledge obtainedin relevant pharmacogenomics studies in determining whether toadminister a therapeutic agent as well as tailoring the dosage and/ortherapeutic regimen of treatment with a therapeutic agent.

Effective delivery of the antisense oligomer to the target nucleic acidis one aspect of treatment. Routes of antisense oligomer deliveryinclude, but are not limited to, various systemic routes, including oraland parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal,and intramuscular, as well as inhalation, transdermal, and topicaldelivery. The antisense oligomer may be aerosolized for delivery. Theappropriate route may be determined by one of skill in the art, asappropriate to the condition of the subject under treatment. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are some non-limiting sites where the antisenseoligomers may be introduced. Direct CNS delivery may be employed, forinstance, intracerebral, intraventricular, or intrathecal administrationmay be used as routes of administration.

In certain embodiments, the antisense oligomers of the disclosure can bedelivered by transdermal methods (e.g., via incorporation of theantisense oligomers into, e.g., emulsions, with such antisense oligomersoptionally packaged into liposomes). Such transdermal andemulsion/liposome-mediated methods of delivery are described fordelivery of antisense oligomers in the art, e.g., in U.S. Pat. No.6,965,025, the contents of which are incorporated in their entirety byreference herein.

In certain embodiments, the antisense oligomers of this disclosure canbe delivered by aerosolization. Advantages to administering medicationsto the lung as an aerosol include: a more rapid onset of action comparedto oral therapy; high local concentration by delivery directly to theairways; needle-free systemic delivery of drugs with poor oralbioavailability; and pain- and needle-free delivery for drugs thatrequire subcutaneous or intravenous injection. Traditional aerosoltherapies with the lung as the target consist of short-actingβ2-adrenergic agonists and long-acting β2-adrenergic agonists (LABA),anticholinergics, inhaled corticosteroids (ICSs), nonsteroidalantiinflammatories, antibiotics and mucolytics. Devices that deliverthese drugs include pressurized metered-dose inhalers (pMDIs), usedeither alone, or attached to spacers, or valved holding chambers (VHCs),breathactuated (BA)-pMDIs, dry powder inhalers (DPIs), jet nebulizers,vibrating mesh nebulizers and soft mist inhalers. Well-establishedtreatment guidelines for the management of asthma and chronicobstructive pulmonary disease (COPD) each recommend inhaled therapy asthe primary route to administer these medications. Treatment guidelinesfor cystic fibrosis (CF) also include recommendations for inhalation ofaerosolized medications.

The antisense oligomers described herein may also be delivered via animplantable device. Design of such a device is an art-recognizedprocess, with, e.g., synthetic implant design described in, e.g., U.S.Pat. No. 6,969,400, the contents of which are incorporated by reference.

Antisense oligomers can be introduced into cells using art-recognizedtechniques (e.g., transfection, electroporation, fusion, liposomes,colloidal polymeric particles and viral and non-viral vectors as well asother means known in the art). The method of delivery selected willdepend at least on the oligomer chemistry, the cells to be treated andthe location of the cells and will be apparent to the skilled artisan.For instance, localization can be achieved by liposomes with specificmarkers on the surface to direct the liposome, direct injection intotissue containing target cells, specific receptor-mediated uptake, orthe like.

As known in the art, antisense oligomers may be delivered using, e.g.,methods involving liposome-mediated uptake, lipid conjugates,polylysine-mediated uptake, nanoparticle-mediated uptake, andreceptor-mediated endocytosis, as well as additional non-endocytic modesof delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (see, e.g., Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44:35-49, incorporated by reference inits entirety).

The antisense oligomers may be administered in any convenient vehicle orcarrier which is physiologically and/or pharmaceutically acceptable.Such a composition may include any of a variety of standardpharmaceutically acceptable carriers employed by those of ordinary skillin the art. Examples include, but are not limited to, saline, phosphatebuffered saline (PBS), water, aqueous ethanol, emulsions, such asoil/water emulsions or triglyceride emulsions, tablets and capsules. Thechoice of suitable physiologically acceptable carrier will varydependent upon the chosen mode of administration. “Pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions

The compounds (e.g., antisense oligomers, antimicrobial agents)described herein may generally be utilized as the free acid or freebase. Alternatively, the compounds of this disclosure may be used in theform of acid or base addition salts. Acid addition salts of the freeamino compounds of the present disclosure may be prepared by methodswell known in the art, and may be formed from organic and inorganicacids. Suitable organic acids include maleic, fumaric, benzoic,ascorbic, succinic, methanesulfonic, acetic, trifluoroacetic, oxalic,propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic,cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, andbenzenesulfonic acids.

Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric,phosphoric, and nitric acids. Base addition salts included those saltsthat form with the carboxylate anion and include salts formed withorganic and inorganic cations such as those chosen from the alkali andalkaline earth metals (for example, lithium, sodium, potassium,magnesium, barium and calcium), as well as the ammonium ion andsubstituted derivatives thereof (for example, dibenzylammonium,benzylammonium, 2-hydroxyethylammonium, and the like). Thus, the term“pharmaceutically acceptable salt” is intended to encompass any and allacceptable salt forms.

In addition, prodrugs are also included within the context of thisdisclosure. Prodrugs are any covalently bonded carriers that release acompound in vivo when such prodrug is administered to a patient.Prodrugs are generally prepared by modifying functional groups in a waysuch that the modification is cleaved, either by routine manipulation orin vivo, yielding the parent compound. Prodrugs include, for example,compounds of this disclosure wherein hydroxy, amine or sulfhydryl groupsare bonded to any group that, when administered to a patient, cleaves toform the hydroxy, amine or sulfhydryl groups. Thus, representativeexamples of prodrugs include (but are not limited to) acetate, formateand benzoate derivatives of alcohol and amine functional groups of theantisense oligomers of the disclosure. Further, in the case of acarboxylic acid (—COOH), esters may be employed, such as methyl esters,ethyl esters, and the like.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligomer into cells (see, e.g., Williams, S. A., Leukemia10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res. 23:119, 1994;Uhlmann et al., antisense oligomers: a new therapeutic principle,Chemical Reviews, Volume 90, No. 4, 25 pages 544-584, 1990; Gregoriadis,G., Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp.287-341, Academic Press, 1979). Hydrogels may also be used as vehiclesfor antisense oligomer administration, for example, as described in WO93/01286. Alternatively, the oligomers may be administered inmicrospheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C. H., J.Biol. Chem. 262:4429-4432, 30 1987). Alternatively, the use ofgas-filled microbubbles complexed with the antisense oligomers canenhance delivery to target tissues, as described in U.S. Pat. No.6,245,747. Sustained release compositions may also be used. These mayinclude semipermeable polymeric matrices in the form of shaped articlessuch as films or microcapsules.

In certain embodiments, the antisense oligomer is administered to amammalian subject, e.g., human or domestic animal, exhibiting thesymptoms of a bacterial infection (e.g., antibiotic resistance or MDRbacterial infection), in a suitable pharmaceutical carrier. In someaspects, the subject is a human subject, e.g., a patient diagnosed ashaving a bacterial infection. In particular embodiments, the antisenseoligomer is contained in a pharmaceutically acceptable carrier, and isdelivered orally. In some embodiments, the antisense oligomer iscontained in a pharmaceutically acceptable carrier, and is deliveredintravenously (i.v.).

In some embodiments, the antisense oligomer is administered in an amountand manner effective to result in a peak blood concentration of at least200-400 nM antisense oligomer. Typically, one or more doses of antisenseoligomer are administered, generally at regular intervals, for a periodof about one to two weeks. Certain doses for oral administration arefrom about 1-1000 mg oligomer per 70 kg. In some cases, doses of greaterthan 1000 mg oligomer/patient may be necessary. For i.v. administration,some doses are from about 0.5 mg to 1000 mg oligomer per 70 kg. Theantisense oligomer may be administered at regular intervals for a shorttime period, e.g., daily for two weeks or less. However, in some casesthe antisense oligomer is administered intermittently over a longerperiod of time. Administration may be followed by, or concurrent with,administration of an antimicrobial (e.g., antibiotic) or othertherapeutic treatment, as described herein. The treatment regimen may beadjusted (dose, frequency, route, etc.) as indicated, based on theresults of immunoassays, other biochemical tests and physiologicalexamination of the subject under treatment.

An effective in vivo treatment regimen using the antisense oligomers ofthe disclosure may vary according to the duration, dose, frequency androute of administration, as well as the condition of the subject undertreatment (i.e., prophylactic administration versus administration inresponse to localized or systemic infection). Accordingly, such in vivotherapy will often include monitoring by tests appropriate to theparticular type of disorder or bacterial infection under treatment, andcorresponding adjustments in the dose or treatment regimen, in order toachieve an optimal therapeutic outcome.

Treatment may be monitored, e.g., by general indicators of disease knownin the art. The efficacy of an in vivo administered antisense oligomerof the disclosure may be determined from biological samples (tissue,blood, urine etc.) taken from a subject prior to, during and subsequentto administration of the antisense oligomer. Assays of such samplesinclude (1) monitoring the presence or absence of heteroduplex formationwith target and non-target sequences, using procedures known to thoseskilled in the art, e.g., an electrophoretic gel mobility assay; (2)monitoring the amount of a mutant mRNA in relation to a reference normalmRNA or protein as determined by standard techniques such as RT-PCR,Northern blotting, ELISA or Western blotting.

III. Combination Therapies

Certain embodiments include combination therapies, for example, theadministration of antisense oligomers in combination with antimicrobialagents such as antibiotics. Combination therapies can be employed, forexample, to increase the sensitivity or susceptibility of a givenbacteria to one or more antimicrobial agents, and thereby improve thetherapeutic outcome (e.g., resolution of the infection). Likewise,certain combination therapies can be employed, for example, to reduce orreverse the antibiotic resistance of a given bacteria to one or moreantimicrobial agents. In particular embodiments, the antisense oligomerreduces the minimum inhibitory concentration (MIC) of an antibioticagainst a bacterium. Also included are pharmaceutical compositions, asdescribed herein, which comprise an antisense oligomer and anantimicrobial agent such as antibiotic.

In some embodiments, the antisense oligomer and the antimicrobial agentare administered separately. In certain embodiments, the antisenseoligomer and the antimicrobial agent are administered sequentially. Insome embodiments, the antisense oligomer and the antimicrobial agent areadministered concurrently, for example, as part of the same or differentpharmaceutical composition.

Examples of antimicrobial agents (e.g., antibiotics) that can beadministered in combination with an antisense oligomer includebeta-lactam antibiotics such as carbapenems, penicillin and penicillinderivatives (or penams), cephalosporins (e.g., Cefacetrile(cephacetrile), Cefadroxil (cefadroxyl; Duricef), Cephalexin (cefalexin;Keflex), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium),Cefaloridine (cephaloradine), Cefalotin (cephalothin; Keflin), Cefapirin(cephapirin; Cefadryl), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin(cephazolin; Ancef, Kefzol), Cefradine (cephradine; Velosef),Cefroxadine, Ceftezole, Cefaclor (Ceclor, Distaclor, Keflor, Raniclor),Cefonicid (Monocid), Cefprozil (cefproxil; Cefzil), Cefuroxime (Zefu,Zinnat, Zinacef, Ceftin, Biofuroksym, Xorimax), Cefuzonam, Cefmetazole,Cefotetan, Cefoxitin, loracarbef (Lorabid); Cephamycins: cefbuperazone,cefmetazole (Zefazone), cefminox, cefotetan (Cefotan), cefoxitin(Mefoxin), Cefotiam (Pansporin), Cefcapene, Cefdaloxime, Cefdinir(Sefdin, Zinir, Omnicef, Kefnir), Cefditoren, Cefetamet, Cefixime (Fixx,Zifi, Suprax), Cefmenoxime, Cefodizime, Cefotaxime (Claforan), Cefovecin(Convenia), Cefpimizole, Cefpodoxime (Vantin, PECEF), Cefteram,Ceftibuten (Cedax), Ceftiofur, Ceftiolene, Ceftizoxime (Cefizox),Ceftriaxone (Rocephin), Cefoperazone (Cefobid), Ceftazidime (Meezat,Fortum, Fortaz), latamoxef (moxalactam), Cefclidine, cefepime(Maxipime), cefluprenam, cefoselis, Cefozopran, Cefpirome (Cefrom),Cefquinome, flomoxef, Ceftobiprole, Ceftaroline, Cefaloram, Cefaparole,Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen,Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftioxide, Cefuracetime),and monobactams (e.g., aztreonam, tigemonam, nocardin A, tabtoxin);aminoglycosides such as tobramycin, gentamicin, kanamycin a, amikacin,dibekacin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E(paromomycin), and streptomycin; tetracyclines such as tetracycline,chlortetracycline, oxytetracycline, demeclocycline, lymecycline,meclocycline, methacycline, minocycline, rolitetracycline, anddoxycyline; sulfonamides such as sulfacetamide, sulfadiazine,sulfadimidine, sulfafurazole, sulfisomidine, sulfadoxine,sulfamethoxazole, sulfamoxole, sulfadimethoxine, sulfamethoxypyridazine,sulfametoxydiazine, sulfadoxine, and sulfametopyrazine; quinolones suchas cinoxacin, nalidixic acid, oxolinic acid (Uroxin), piromidic acid(Panacid), pipemidic acid (Dolcol) rosoxacin (Eradacil), ciprofloxacin(Alcipro, Ciprobay, Cipro, Ciproxin, ultracipro), enoxacin (Enroxil,Penetrex), fleroxacin (Megalone, Roquinol), lomefloxacin (Maxaquin),nadifloxacin (Acuatim, Nadoxin, Nadixa), norfloxacin (Lexinor, Noroxin,Quinabic, Janacin), ofloxacin (Floxin, Oxaldin, Tarivid), pefloxacin(Peflacine), rufloxacin (Uroflox), balofloxacin (Baloxin), grepafloxacin(Raxar), levofloxacin (Cravit, Levaquin, Tavanic), pazufloxacin (Pasil,Pazucross), sparfloxacin (Zagam), temafloxacin (Omniflox), tosufloxacin(Ozex, Tosacin), clinafloxacin, gatifloxacin (Zigat, Tequin)(Zymar-opth.), gemifloxacin (Factive), moxifloxacin (Acflox Woodward,Avelox, Vigamox, sitafloxacin (Gracevit), trovafloxacin (Trovan),prulifloxacin (Quisnon); oxazolidinones such as eperezolid, linezolid,posizolid, radezolid, ranbezolid, sutezolid, and tedizolid; polymyxinssuch as polysporin, neosporin, polymyxin B, polymyxin E (colistin);rifamycins such as rifampicin or rifampin, rifabutin, rifapentine, andrifaximin; lipiarmycins such as fidaxomicin; macrolides such asazithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, telithromycin, carbomycin A, josamycin, kitasamycin,midecamycin/midecamycin acetate, oleandomycin, solithromycin,spiramycin, and troleandomycin; lincosamides such as lincomycin,clindamycin, and pirlimycin; cyclic lipopeptides such as daptomycin;glycopeptides such as vancomycin and teichoplanin; glycylcyclines suchas tigecycline. Thus, any one or more of the foregoing antibiotics canbe combined with any of the antisense oligomers described herein, forthe treatment of any of the bacteria described herein.

In some embodiments, the antimicrobial agent is a beta-lactamantibiotic, as described herein. In certain of these and relatedembodiments, the bacterium comprises or expresses a beta-lactamase suchas NDM-1, and the antisense oligomer is targeted against thebeta-lactamase. In particular embodiments, the antimicrobial agent is acarbapenem. Examples of carbapenems include meropenem, imipenem,ertapenem, doripenem, panipenem, biapenem, razupenem, tebipenem,lenapenem, and tomopenem. In certain of these and related embodiments,the bacterium comprises or expresses a carbapenemase such as NDM-1, andthe antisense oligomer is targeted against the carbapenemase. Inspecific embodiments, the bacterium is Escherichia coli, Acinetobacterbaumannii, or Klebsiella pneumoniae.

In some embodiments, the antimicrobial agent is an aminoglycoside suchas tobramycin or gentamicin or a tetracycline, as described herein. Insome of these and related embodiments, the bacterium comprises orexpresses the antibiotic resistance gene adeA, and the antisenseoligomer is targeted against the antibiotic resistance gene. In specificembodiments, the bacterium is Escherichia coli, Acinetobacter baumannii,or Klebsiella pneumoniae.

In certain embodiments, the antimicrobial agent includes one or more ofceftazidime, doxycycline, piperacillin, meropenem, chloramphenicol,and/or co-trimoxazole (trimethoprim/sulfamethoxazole). In some of theseand related embodiments, the bacterium is a Burkholderia species thatcomprises or expresses one or more biofilm formation genes such as cepI,cepR, and/or suhB, and the antisense oligomer is targeted against thebiofilm formation gene(s). In specific embodiments, the bacterium isBurkholderia cepacia or a Burkholderia cepacia complex. In specificembodiments, the subject is immunocompromised and has an underlying lungdisease, such as cystic fibrosis (CF) or chronic granulomatous disease(CGD).

In certain embodiments, the antimicrobial agent includes one or more ofceftazidime, doxycycline, piperacillin, minocycline, meropenem,chloramphenicol, and/or co-trimoxazole (trimethoprim/sulfamethoxazole).In some of these and related embodiments, the bacterium is aBurkholderia species that comprises or expresses one or more genesassociated with fatty acid biosynthesis such as acpP, and the antisenseoligomer is targeted against the gene(s) encoding an acyl carrierprotein. In specific embodiments, the bacterium is Burkholderia cepaciaor a Burkholderia cepacia complex. In specific embodiments, the subjectis immunocompromised and has an underlying lung disease, such as cysticfibrosis (CF) or chronic granulomatous disease (CGD).

In some embodiments, the antisense oligomer increases the sensitivity ofa given bacteria to the antimicrobial agent, relative to theantimicrobial agent alone. For example, in certain embodiments, theantisense oligomer increases the sensitivity of the bacterium to theantimicrobial agent by increasing the bactericidal (cell-killing) and/orbacteriostatic (growth-slowing) activity of the antimicrobial agentagainst the bacterium being targeted, relative to the antimicrobialagent alone. In particular embodiments, the antisense increases thesensitivity by about or at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, or 1000% or more (including allintegers and ranges in between), relative to the antimicrobial agentalone, or by about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-foldor more (including all integers and ranges in between), relative to theantimicrobial agent alone.

In some embodiments, the antisense oligomer reduces the minimuminhibitory concentration (MIC) of an antimicrobial agent against thebacterium being targeted, relative to the antimicrobial agent alone. The“minimum inhibitory concentration” or “MIC” refers to the lowestconcentration of an antimicrobial agent that will inhibit the visiblegrowth of a microorganism after overnight (in vitro) incubation. Minimuminhibitory concentrations are important in diagnostic laboratories toconfirm resistance of microorganisms to an antimicrobial agent and alsoto monitor the activity of new antimicrobial agents. The MIC isgenerally regarded as the most basic laboratory measurement of theactivity of an antimicrobial agent against a bacterial organism. Thus,in certain embodiments, the oligomer reduces the minimum inhibitoryconcentration (MIC) of an antimicrobial agent against the bacterium byat least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, or 1000% or more (including all integers and ranges inbetween), relative to the antimicrobial agent alone. In certainembodiments, the oligomer reduces the minimum inhibitory concentration(MIC) of an antimicrobial agent against the bacterium by about or atleast about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold or more (including allintegers and ranges in between), relative to the antimicrobial agentalone.

In some embodiments, the antisense oligomer that increases thesensitivity or reduces the MIC is targeted against NDM-1, the bacteriumis Escherichia coli, Acinetobacter baumannii, or Klebsiella pneumoniaethat comprises or expresses NDM-1, and the antimicrobial agent is acarbapenem such as meropenem, imipenem, ertapenem, doripenem, panipenem,biapenem, razupenem, tebipenem, lenapenem, or tomopenem.

In particular embodiments, the antisense oligomer that increases thesensitivity or reduces the MIC is targeted against adeA, the bacteriumis Escherichia coli, Acinetobacter baumannii, or Klebsiella pneumoniaethat comprises or expresses adeA, and the antimicrobial agent is anaminoglycoside antibiotic (e.g., tobramycin, gentamicin, kanamycin a,amikacin, dibekacin, sisomicin, netilmicin, neomycin B, neomycin C,neomycin E (paromomycin), streptomycin), a tetracycline antibiotic(e.g., tetracycline, chlortetracycline, oxytetracycline, demeclocycline,lymecycline, meclocycline, methacycline, minocycline, rolitetracycline,doxycyline), or a β-lactam antibiotic (e.g., carbapenem, penicillinderivative (penam), cephalosporin (cephem), monobactam).

In particular embodiments, the antisense oligomer that increases thesensitivity or reduces the MIC is targeted against cepI, the bacteriumis a Burkholderia species, for example, Burkholderia cepacia (complex)or a sub-species thereof (e.g., Burkholderia cenocepacia, Burkholderiamultivorans, Burkholderia vietnamiensis, Burkholderia stabilis,Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia dolosa,Burkholderia ambifaria), which comprises or expresses cepI, and theantimicrobial agent is selected from one or more of ceftazidime,doxycycline, piperacillin, meropenem, chloramphenicol, andco-trimoxazole (trimethoprim/sulfamethoxazole).

In particular embodiments, the antisense oligomer that increases thesensitivity or reduces the MIC is targeted against suhB, the bacteriumis a Burkholderia species, for example, Burkholderia cepacia (complex)or a sub-species thereof (e.g., Burkholderia cenocepacia, Burkholderiamultivorans, Burkholderia vietnamiensis, Burkholderia stabilis,Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia dolosa,Burkholderia ambifaria), which comprises or expresses suhB, and theantimicrobial agent is selected from one or more of ceftazidime,doxycycline, piperacillin, meropenem, chloramphenicol, andco-trimoxazole (trimethoprim/sulfamethoxazole).

In particular embodiments, the antisense oligomer that increases thesensitivity or reduces the MIC is targeted against acpP, the bacteriumis a Burkholderia species, for example, Burkholderia cepacia (complex)or a sub-species thereof (e.g., Burkholderia cenocepacia, Burkholderiamultivorans, Burkholderia vietnamiensis, Burkholderia stabilis,Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia dolosa,Burkholderia ambifaria), which comprises or expresses acpP, and theantimicrobial agent is selected from one or more of ceftazidime,doxycycline, piperacillin, minocycline, meropenem, chloramphenicol, andco-trimoxazole (trimethoprim/sulfamethoxazole).

IV. Treatment Monitoring Methods

The efficacy of a given therapeutic regimen involving the methodsdescribed herein may be monitored, for example, by general indicators ofbacterial infection, such as complete blood count (CBC), nucleic aciddetection methods, immunodiagnostic tests, or bacterial culture.

In some aspects, identification and monitoring of bacterial infectioninvolves one or more of (1) nucleic acid detection methods, (2)serological detection methods, i.e., conventional immunoassay, (3)culture methods, and (4) biochemical methods. Such methods may bequalitative or quantitative.

Nucleic acid probes may be designed based on publicly availablebacterial nucleic acid sequences, and used to detect target genes ormetabolites (i.e., toxins) indicative of bacterial infection, which maybe specific to a particular bacterial type, e.g., a particular speciesor strain, or common to more than one species or type of bacteria (i.e.,Gram positive or Gram negative bacteria). Nucleic amplification tests(e.g., PCR) may also be used in such detection methods.

Serological identification may be accomplished using a bacterial sampleor culture isolated from a biological specimen, e.g., stool, urine,cerebrospinal fluid, blood, etc. Immunoassay for the detection ofbacteria is generally carried out by methods routinely employed by thoseof skill in the art, e.g., ELISA or Western blot. In addition,monoclonal antibodies specific to particular bacterial strains orspecies are often commercially available.

Culture methods may be used to isolate and identify particular types ofbacteria, by employing techniques including, but not limited to, aerobicversus anaerobic culture, growth and morphology under various cultureconditions. Exemplary biochemical tests include Gram stain (Gram, 1884;Gram positive bacteria stain dark blue, and Gram negative stain red),enzymatic analyses, and phage typing.

It will be understood that the exact nature of such diagnostic, andquantitative tests as well as other physiological factors indicative ofbacterial infection will vary dependent upon the bacterial target, thecondition being treated and whether the treatment is prophylactic ortherapeutic.

In cases where the subject has been diagnosed as having a particulartype of bacterial infection, the status of the bacterial infection isalso monitored using diagnostic techniques typically used by those ofskill in the art to monitor the particular type of bacterial infectionunder treatment.

The PMO or PPMO treatment regimen may be adjusted (dose, frequency,route, etc.), as indicated, based on the results of immunoassays, otherbiochemical tests and physiological examination of the subject undertreatment.

From the foregoing, it will be appreciated how various objects andfeatures of the disclosure are met. The method provides an improvementin therapy against bacterial infection, for example, multi-drugresistant (MDR) bacteria and/or biofilm-forming bacteria, usinganti-virulence antisense oligomers to achieve enhanced cell uptake andanti-bacterial action. As a result, drug therapy is more effective andless expensive, both in terms of cost and amount of compound required.

One exemplary of the disclosure is that compounds effective againstvirtually any pathogenic bacterial can be readily designed and tested,e.g., for rapid response against new drug-resistant strains.

The following examples are intended to illustrate but not to limit thedisclosure. Each of the patent and non-patent references referred toherein is incorporated by reference in its entirety.

EXAMPLES Example 1 Activity of PPMOs Targeted Against adeA

A cell-penetrating peptide-conjugated phosphorodiamidate morpholinooligomer (PPMOs) targeted against the resistance-nodulation-celldivision (RND)-type multidrug efflux pump subunit adeA (adeA) wasprepared and tested for the ability to reduce the minimum inhibitoryconcentration (MIC) of various antibiotics against adeA-expressingAcinetobacter baumanii.

The adeA-targeted PPMO has the following sequence: ATACTGTCCAA (SEQ IDNO: 15; PPMO#5). The PPMO was conjugated at its 3′-end to the C-terminalβ-alanine residue of (RXR)₄XB (SEQ ID NO: 40).

The MIC of the antibiotics gentamicin, tobramycin, and tetracycline wasmeasured using the microdilution method of the Clinical LaboratoryStandards Institute in a 96-well microtiter plate format. Multiple,identical dilution series of each antibiotic were included on eachmicrotiter plate. In each dilution series of antibiotic, a fixed amountof PPMO was added. Each dilution series of antibiotic included adifferent concentration of PPMO.

The results are shown in FIGS. 2-4. These figures show that treatment ofadeA (efflux pump)-expressing Acinetobacter baumanii with theadeA-targeted PPMO significantly reduced the MIC of gentamicin (FIG. 2),tobramycin (FIG. 3), and tetracycline (FIG. 4), each in a concentrationdependent manner.

Example 2 Activity of PPMOs Targeted Against NDM-1

Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)targeted against the New Delhi metallo-beta-lactamase (NDM-1) wereprepared and tested for the ability to reduce the minimum inhibitoryconcentration (MIC) of meropenem against NDM-1-expressing Acinetobacterbaumanii and E. coli.

The NDM-1 targeted PPMOs have the following sequences: TCAAGTTTTCC (SEQID NO: 11; PPMO#1); TCCTTTTATTC (SEQ ID NO: 12; PPMO#2); CCATCAAGTTT(SEQ ID NO: 13; PPMO#3); and GGCAATTCCAT (SEQ ID NO: 14; PPMO#4). Eachof the PPMOs was conjugated at its 3′-end to the C-terminal β-alanineresidue of (RXR)₄XB (SEQ ID NO: 40).

The MIC of meropenem was measured using the microdilution method of theClinical Laboratory Standards Institute in a 96-well microtiter plateformat. Multiple, identical dilution series of meropenem were includedon each microtiter plate. In each dilution series of meropenem, a fixedamount of PPMO was added. Each dilution series of meropenem included adifferent concentration of PPMO.

As shown in FIGS. 5A-5B and 6, NDM-1-targeted PPMOs reduced the MIC ofmeropenem from about 8 to 32-fold, depending on the bacterium. Thesefigures show that treatment of NDM-1-expressing Acinetobacter baumanii(FIG. 5A) and NDM-1-expressing E. coli (FIG. 6) with NDM-1-targetedPPMOs significantly reduced the MIC of meropenem in a concentrationdependent manner. FIG. 5B shows that the NDM-1 targeted PPMO andmeropenem synergistically reduced the number of colony-forming units(CFUs) of NDM-1-expressing Acinetobacter baumanii. Meropenemaseenzymatic activity in the periplasm of PPMO-treated cells was thusobserved to be inversely proportional to the amount of PPMO added.

Similar effects were shown for Klebsiella pneuomoniae; at aconcentration of 8 μM, the most effective NDM-1-targeted PPMO reducedthe MIC of meropenem from about 64 μM to about 4 μM (data not shown).

Thus, the NDM-1-targeted PPMOs silenced expression of NDM-1 and reducedthe MIC of meropenem to susceptible concentrations in threemultidrug-resistant pathogens.

Example 3 Activity of PPMOs Targeted Against Biofilm and Acyl CarrierProtein Genes

Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)targeted against the biofilm formation genes suhB and cepI and the acylcarrier protein acpP gene were prepared and tested for the ability toreduce biofilm formation and to break down established biofilm inBurkholderia cenocepacia J2315.

The suhB-targeted PPMOs have the following sequences: ATGCATGAGCC (SEQID NO: 23; PPMO#13); and GGATGCATGAG (SEQ ID NO: 24; PPMO#14).

The cepI-targeted PPMOs have the following sequences: AAGGTCTGCAT (SEQID NO: 16; PPMO#6); TCGGATCTGTG (SEQ ID NO: 17; PPMO#7); CATGGATGTCC(SEQ ID NO: 18; PPMO#8); CGTGAACGAAG (SEQ ID NO: 19; PPMO#9);CGTGTGGCAAC (SEQ ID NO: 20; PPMO#10); GCCCGAGATCC (SEQ ID NO: 21;PPMO#11); and CTTTCGTTCGC (SEQ ID NO: 22; PPMO#12).

Each of the suhB-targeted and cepI-targeted PPMOs was conjugated at its3′-end to the C-terminal β-alanine residue of (RFF)₃RXB (SEQ ID NO: 41).

The acpP-targeted PPMOs have the sequences and peptide conjugations asshown in Table 3C (PPMO #s 15-29).

Biofilms were formed in 150 μl cultures of Burkholderia cenocepaciaJ2315, using 96-well polystyrene microtiter plates. To test the abilityof PPMOs to reduce biofilm formation, PPMOs (1-10 μM) were added tobacterial cultures prior to biofilm formation and incubated withbacteria for 48 hours. To test the ability of PPMOs to reduceestablished biofilms, bacterial cultures were grown for 48 hours andallowed to form biofilms prior to addition of PPMOs (1-10 μM), and thenincubated for an additional 48 hours in the presence of PPMOs. Foranalysis, the liquid cultures were removed and the biofilms that adheredto the microtiter plate were stained with crystal violet. The amount ofcrystal violet stain in the biofilm was measured, and found to beproportional to the amount of biofilm. Confocal laser scanningmicroscopy (CLSM) and dsRed expressing Burkholderia cenocepacia J2315were used to visualize biofilm structural changes (see FIGS. 8A-8C).

As shown in FIGS. 7A-7B, treatment of biofilm-forming Burkholderia withPPMOs targeted against acpP, suhB or cepI not only disrupted theformation of biofilm (7A; PPMOs were added prior to biofilm formationand incubated for 48 hours) but also broke down established biofilms(7B; biofilm was grown for 48 hours prior to 48-hour incubation withPPMOs). A 10 μM concentration of acpP-targeted PPMO reduced biofilmformation by about 45% and reduced existing biofilm by about 50%. A 10μM concentration of cepI-targeted PPMO reduced biofilm formation byabout 52% and reduced existing biofilm by about 65%. A 10 μMconcentration of suhB-targeted PPMO reduced biofilm formation by about40% and reduced existing biofilm by about 42%. Thus, when biofilms werevisualized with CLSM there was a dramatic reduction in biofilm formationin the presence of cepI-targeted and suhB-targeted PPMOs.

Example 4 PPMOs Act Synergistically with Antibiotics to Reduce BacterialGrowth in Established Biofilms

PPMOs targeted against the biofilm formation gene cepI were prepared andtested for the ability to reduce bacterial growth in establishedbiofilms in combination with the aminoglycoside antibiotic Tobramycin inBurkholderia cenocepacia J2315.

The cepI-targeted PPMOs have the following sequences: PPMO#s 6-12 inTable 3B.

Each of the PPMOs was conjugated at the 3′ terminus with (RFF)₃RXB (SEQID NO: 41).

To test the ability of cepI PPMOs and Tobramycin to reduce bacterialgrowth in established biofilms, bacterial cultures were grown for 48hours and allowed to form biofilms prior to addition of PPMOs or PPMOand Tobramycin, and then incubated for an additional 48 hours in thepresence of no PPMOs, scrambled control PPMO, scrambled control PPMOwith Tobramycin at either 64 μg/mL or 128 μg/mL, cepI PPMO, and cepIPPMO with Tobramycin at either 64 μg/mL or 128 μg/mL. Bacterial growthwas measured as CFU/mL.

As shown in FIG. 9, cepI PPMO alone or scrambled PPMO with Tobramycinwas able to inhibit bacterial growth significantly about 2 logs comparedto the untreated biofilm. cepI PPMO in combination with Tobramycin,however, was further able to significantly inhibit bacterial growth onthe established biofilm, and at the higher concentration of Tobramycin(128 μg/mL), was able to reduce the bacterial CFU/mL by another logcompared to cepI PPMO alone.

Example 5 PPMOs Inhibit Members of the Bcc

A variety of Bcc isolates were tested, including clinical isolatesobtained from various body sites with varying levels of antibioticresistance. The strain bank included 39 isolates that comprised the mostfrequently encountered species that have been reported to cause humandisease. As shown in FIG. 10, 6 PPMOs (PPMO#s 19-23 and 25) achievedIC₇₃ values of 8 μM or less. All 6 of these PPMOs targeted AcpP (an acylcarrier protein associated with fatty acid biosynthesis). Differences inthe 6 PPMOs related to alternative positioning sites on the target mRNA.The most potent PPMO (PPMO#19) had an IC₇₃ of 4 μM. acpP PPMO targetingsequences are listed in Table 3C.

Example 6 PPMOs are Bactericidal in Bcc

Many members of the Bcc are intrinsically antibiotic resistant makingtreatment difficult. B. cenocepacia is one of the most common speciesencountered by cystic fibrosis (CF) patients. Two different isolates ofB. cenocepacia were incubated for 24 hours in the presence or absence ofdifferent acpP PPMOs (FIG. 11).

The acpP-targeted PPMOs have the following sequences: GTCCATTACCC(PPMO#15; SEQ ID NO: 25); CCATTACCCCT (PPMO#19; SEQ ID NO: 27); andTTGTCCATTAC (PPMO#22; SEQ ID NO: 30).

Each of the PPMOs was conjugated at its 5′-end to the C-terminalβ-alanine residue of (RFF)₃RXB (SEQ ID NO: 41).

In B. cenocepacia K56-2 (a genome-sequenced clinical CF isolate; PanelA) all three PPMOs caused a significant reduction of growth with onePPMO (PPMO#19; SEQ ID NO: 27; 16 μM) causing >3-log reduction of growthcompared to the starting inoculum, a strong bactericidal effect.Importantly, this effect was seen even in pan-resistant strains of B.cenocepacia (HI4277, a pan-resistant outbreak isolate from CF patients;FIG. 11, Panel B). Even though this strain demonstrated resistance toall traditional antibiotics, PPMO#19 was bactericidal. The MIC ofPPMO#19 was 8 μM in HI4277 (FIG. 10), illustrating that PPMOs' abilityto inhibit growth is not dependent on the underlying level of antibioticresistance in any particular strain, an important finding with positiveimplications for this approach.

Example 7 PPMOs Inhibit Bcc Growth in Sputum

Chronic infections in the CF patient usually manifest in the lung. Inaddition, members of the Bcc and P. aeruginosa are known to formbiofilms. These biofilms and the thick sputum formed by CF patientsmakes treatment with antibiotics particularly difficult and thesepathogens become virtually impossible to completely eradicate from thelung environment.

PPMOs were tested to determine whether they retained their activity insputum. Using a well-described method for making “artificial CF sputum,”experiments were conducted to see whether a PPMO could reduce the burdenof Bcc in this environment (FIG. 12). B. cenocepacia K56-2 was incubatedalone or in the presence of either a scrambled-sequence (Scr) placeboPPMO or acpP PPMO (PPMO#15). The acpP-targeted PPMO has the followingsequence: GTCCATTACCC (PPMO#15; SEQ ID NO: 25). The PPMO was conjugatedat its 5′ end to the C-terminal β-alanine residue of (RFF)₃RXB (SEQ IDNO: 41). Media or PPMO was dosed at 2, 8 and 12 hours. Samples wereplated at 24 hours and CFU/ml was determined. The acpP PPMO was able toreduce the organism burden (both at 10 and 20 μM dosing). At 10 μMdosing, there was an approximately 2-log reduction in CFU/ml by 24-hourscompared to no treatment control. At 20 μM dosing, there was a >3-logreduction seen. This reduction was apparent as early as 8 hours in the20 μM group and 12 hours in the 10 μM group. These experiments indicatethat PPMOs remain active even in the thick viscous sputum that is seenin CF patients. This is the first time that activity of PPMOs has beentested in sputum.

Example 8 acpP PPMOs can Both Prevent Biofilm Formation and DeconstructExisting Biofilms

The formation of biofilm is a significant virulence trait and isutilized by both P. aeruginosa and the Bcc. Bcc biofilms were grown andPPMOs were tested for their ability to both prevent biofilm formationand to deconstruct existing biofilms. B. cenocepacia J2315 (a genomesequenced, epidemic CF isolate) was grown utilizing MBEC biofilm assayplates which contain a peg that grows reproducible biofilms from day today. J2315 was grown for 48 hours in the presence of either acpP PPMO(10 μM), scrambled PPMO (10 μM), peptide or media alone (FIG. 13). TheacpP-targeted PPMO (PPMO#19) has the following sequence: CCATTACCCCT(SEQ ID NO: 27). The PPMO was conjugated at its 5′ end to thebeta-alanine residue of (RFF)₃RXB (SEQ ID NO: 41). Biofilm productionwas measured utilizing a crystal violet method. As can be seen, the acpPPPMO reduced biofilm formation by >50% compared to controls. The acpPPPMO was then tested to see if it could break down an existing biofilm.Biofilms were grown for 48 hours and then the mature biofilm pegs weretransferred to a fresh plate with media alone (FIG. 14A), scrambledcontrol PPMO at 10 μM (FIG. 14B) or acpP PPMO at 10 μM (FIG. 14C). Theplates were incubated for 48 hours more and biofilm formation wasmeasure both by crystal violet as well as by confocal microscopy. Asmeasured by a fluorescent-red expressing J2315 strain, the acpP PPMOsignificantly reduced the amount of biofilm present both by confocalmicroscopy and crystal violet measurements (FIG. 14C). The ability ofPPMOs (designed against essential gene targets) to break down existingbiofilms is a novel and critically important finding.

Example 9 Aerosol Delivery of PPMO Reduces Burden of B. multivorans in aPulmonary Infection Model

Delivering PPMOs directly to the lung in the setting of chronicpulmonary infections would be useful. Chronic granulomatous disease(CGD) mice were used as a Bcc infection model. These mice developsignificant morbidity and mortality when infected with various Bccstrains. Mice were infected intranasally with a clinical B. multivoransisolate (FIG. 15). An Aerogen nebulizer was used to deliver eitherscrambled (Scr) PPMO (300 μg) or acpP PPMO (PPMO#15, 300 μg or 30 μg) asa one-time dose 6 hours post-infection. The acpP-targeted PPMO has thefollowing sequence: GTCCATTACCC (SEQ ID NO: 25). The PPMO was conjugatedat its 5′ end to the C-terminal β-alanine residue of (RFF)₃RXB (SEQ IDNO: 41). Mice were euthanized 24 hours after infection and lung burdenwas determined. The single 300 μg dose of the acpP PPMO reduced the lungburden by 93% and was a statistically significant decrease. Aerosoldelivery of PPMOs is a viable therapeutic strategy and importantly, thisis the first time that nebulized delivery of Bcc PPMOs has beenattempted.

The invention claimed is:
 1. An antisense morpholino oligomer of formula(I):

or a pharmaceutically acceptable salt thereof, where each Nu is anucleobase which taken together forms a targeting sequence; X is aninteger from 9 to 38; T is selected from OH and a moiety of the formula:

where each R⁴ is independently C₁-C₆ alkyl, and R⁵ is selected from anelectron pair and H, and R⁶ is selected from OH, —N(R⁷)CH₂C(O)NH₂, and amoiety of the formula:

where: R⁷ is selected from H and C₁-C₆ alkyl, and R⁸ is selected from G,—C(O)—R⁹OH, acyl, trityl, and 4-methoxytrityl, where: R⁹ is of theformula —(O-alkyl)_(y)- wherein y is an integer from 3 to 10 and each ofthe y alkyl groups is independently selected from C₂-C₆ alkyl; eachinstance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is independently C₁-C₆alkyl, and R¹¹ is selected from an electron pair and H; R² is selectedfrom H, G, acyl, trityl, 4-methoxytrityl, benzoyl, stearoyl, and amoiety of the formula:

where L is selected from —C(O)(CH₂)₆C(O)— and —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,and each R¹² is of the formula —(CH₂)₂OC(O)N(R¹⁴)₂ wherein each R¹⁴ isof the formula —(CH₂)₆NHC(═NH)NH₂; and R³ is selected from an electronpair, H, and C₁-C₆ alkyl, wherein G is a cell penetrating peptide(“CPP”) and linker moiety selected from —C(O)(CH₂)₅NH—CPP,—C(O)(CH₂)₂NH—CPP, —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, and —C(O)CH₂NH—CPP, orG is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus, with the proviso that only one instance of G ispresent, wherein the targeting sequence specifically hybridizes to abacterial mRNA target sequence that encodes a virulence factor, andwherein the targeting sequence is selected from:a) SEQ ID NO: 11 (TCA AGT TTT CC); b) SEQ ID NO: 12 (TCC TTT TAT TC);c) SEQ ID NO: 13 (CCA TCA AGT TT); d) SEQ ID NO: 14 (GGC AAT TCC AT);e) SEQ ID NO: 15 (ATA CTG TCC AA);

wherein X is 9, and thymine bases (T) may be uracil bases(U).
 2. Theantisense morpholino oligomer of claim 1, wherein T is selected from:


3. The antisense morpholino oligomer of claim 2, wherein R² is selectedfrom H, G, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.
 4. Theantisense morpholino oligomer of claim 1, wherein T is selected from:

and R² is G.
 5. The antisense morpholino oligomer of claim 1, wherein Tis of the formula:

R⁶ is of the formula:

and R² is G.
 6. The antisense morpholino oligomer of claim 1, wherein Tis of the formula:

and R² is G.
 7. The antisense morpholino oligomer of claim 1, wherein Tis of the formula:


8. The antisense morpholino oligomer according to claim 7, wherein R² isselected from H, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.9. The antisense morpholino oligomer according to claim 8, wherein atleast one instance of R¹ is —N(CH₃)₂.
 10. The antisense morpholinooligomer of claim 9, wherein each R¹ is —N(CH₃)₂.
 11. The antisensemorpholino oligomer according to claim 1, wherein the CPP is selectedfrom:

wherein R^(a) is selected from H, acetyl, benzoyl, and stearoyl.
 12. Theantisense morpholino oligomer according to claim 1, wherein G isselected from:

wherein R^(a) is selected from H, acetyl, benzoyl, and stearoyl.
 13. Anantisense morpholino oligomer of formula (VII) selected from:

or a pharmaceutically acceptable salt of any of the foregoing, whereinR^(a) is H or acetyl, each Nu is a nucleobase which taken together formsa targeting sequence, and the targeting sequence is selected from: f)SEQ ID NO: 11 (TCA AGT TTT CC); g) SEQ ID NO: 12 (TCC TTT TAT TC); h)SEQ ID NO: 13 (CCA TCA AGT TT); i) SEQ ID NO: 14 (GGC AAT TCC AT); j)SEQ ID NO: 15 (ATA CTG TCC AA); wherein X is 9, and thymine bases (T)may be uracil bases(U).
 14. A pharmaceutical composition, comprising apharmaceutically acceptable carrier and an antisense morpholino oligomerof formula (I):

or a pharmaceutically acceptable salt thereof, where each Nu is anucleobase which taken together forms a targeting sequence; X is aninteger from 9 to 38; T is selected from OH and a moiety of the formula:

where each R⁴ is independently C₁-C₆ alkyl, and R⁵ is selected from anelectron pair and H, and R⁶ is selected from OH, —N(R⁷)CH₂C(O)NH₂, and amoiety of the formula:

where: R⁷ is selected from H and C₁-C₆ alkyl, and R⁸ is selected from G,—C(O)—R⁹OH, acyl, trityl, and 4-methoxytrityl, where: R⁹ is of theformula —(O-alkyl)_(y)- wherein y is an integer from 3 to 10 and each ofthe y alkyl groups is independently selected from C₂-C₆ alkyl; eachinstance of R¹ is —N(R¹⁰)₂R¹¹ wherein each R¹⁰ is independently C₁-C₆alkyl, and R¹¹ is selected from an electron pair and H; R² is selectedfrom H, G, acyl, trityl, 4-methoxytrityl, benzoyl, stearoyl, and amoiety of the formula:

where L is selected from —C(O)(CH₂)₆C(O)— and —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,and each R¹² is of the formula —(CH₂)₂OC(O)N(R¹⁴)₂ wherein each R¹⁴ isof the formula —(CH₂)₆NHC(═NH)NH₂; and R³ is selected from an electronpair, H, and C₁-C₆ alkyl, wherein G is a cell penetrating peptide(“CPP”) and linker moiety selected from —C(O)(CH₂)₅NH—CPP,—C(O)(CH₂)₂NH—CPP, —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, and —C(O)CH₂NH—CPP, orG is of the formula:

wherein the CPP is attached to the linker moiety by an amide bond at theCPP carboxy terminus, with the proviso that only one instance of G ispresent, wherein the targeting sequence specifically hybridizes to abacterial mRNA target sequence that encodes a virulence factor, andwherein the targeting sequence is selected from: k) SEQ ID NO: 11 (TCAAGT TTT CC); l) SEQ ID NO: 12 (TCC TTT TAT TC); m) SEQ ID NO: 13 (CCATCA AGT TT); n) SEQ ID NO: 14 (GGC AAT TCC AT); o) SEQ ID NO: 15 (ATACTG TCC AA); wherein X is 9, and thymine bases (T) may be uracilbases(U).
 15. The antisense oligomer of claim 13, wherein the antisenseoligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 11 (TCA AGTTTT CC) wherein X is
 9. 16. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 12 (TCC TTT TAT TC)wherein X is
 9. 17. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 13 (CCA TCA AGT TT)wherein X is
 9. 18. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 14 (GGC AAT TCC AT)wherein X is
 9. 19. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 11 (TCA AGTTTT CC) wherein X is
 9. 20. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 12 (TCC TTT TAT TC)wherein X is
 9. 21. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 13 (CCA TCA AGT TT)wherein X is
 9. 22. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 14 (GGC AATTCC AT) wherein X is
 9. 23. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 11 (TCA AGT TTT CC)wherein X is
 9. 24. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 12 (TCC TTT TAT TC)wherein X is
 9. 25. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 13 (CCA TCA AGT TT)wherein X is
 9. 26. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 14 (GGC AAT TCC AT)wherein X is
 9. 27. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 11 (TCA AGT TTT CC)wherein X is
 9. 28. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 12 (TCC TTT TAT TC)wherein X is
 9. 29. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 13 (CCA TCAAGT TT) wherein X is
 9. 30. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein W is H or acetyl,and the targeting sequence consists of SEQ ID NO: 14 (GGC AAT TCC AT)wherein X is
 9. 31. The antisense oligomer of claim 13, wherein theantisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 11 (TCA AGTTTT CC) wherein X is
 9. 32. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 12 (TCC TTTTAT TC) wherein X is
 9. 33. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 13 (CCA TCAAGT TT) wherein X is
 9. 34. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 14 (GGC AATTCC AT) wherein X is
 9. 35. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 11 (TCA AGTTTT CC) wherein X is
 9. 36. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 12 (TCC TTTTAT TC) wherein X is
 9. 37. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 13 (CCA TCAAGT TT) wherein X is
 9. 38. The antisense oligomer of claim 13, whereinthe antisense oligomer is:

or a pharmaceutically acceptable salt thereof, wherein R^(a) is H oracetyl, and the targeting sequence consists of SEQ ID NO: 14 (GGC AATTCC AT) wherein X is 9.